Abrogen polypeptides, nucleic acids encoding them and methods for using them to inhibit angiogenesis

ABSTRACT

The invention relates to abrogen polypeptides and nucleic acids that encode them. In general, the abrogen polypeptides comprise the kringle domain from, for example, urokinase plasminogen activator. Abrogen polypeptides can be used to inhibit endothelial cell activation and/or proliferation and can inhibit endothelial cells activated or induced by both bFGF and VEGF. The invention also encompasses methods to produce polypeptides that possess abrogen activity as well as method for using these polypeptides.

FIELD OF THE INVENTION AND INTRODUCTION

[0001] The present invention relates to novel nucleic acids encodingnovel amino acid fragments of polypeptides, called abrogens. The presentinvention also relates to novel, potent in vitro and in vivo inhibitorsof endothelial cells proliferation, and compositions of them and uses ofthem. The present invention further provides methods that are effectivefor modulating angiogenesis and inhibiting unwanted angiogenesis.Therefore, polypeptides according to the present invention are usefulfor treating and/or preventing cancer, tumor growth, or other angiogenicdependent or angiogenic associated diseases.

BACKGROUND OF THE INVENTION

[0002] Angiogenesis is the generation of new blood vessels frompreexisting vessels into a tissue or organ. Angiogenesis is required andnormally observed under normal physiological conditions, such as forexample, for wound healing, fetal and embryonic development, for femalereproduction, i.e., formation of the corpus luteum, endometrium andplacenta, organ formation, tissue regeneration and remodeling (Risau Wet al., Nature, 1997, 386, 671-674).

[0003] Angiogenesis begins with local degradation of the basementmembrane of capillaries, followed by invasion of stroma by underlyingendothelial cells in the direction of an angiogenic stimulus. Subsequentto migration, endothelial cells proliferate at the leading edge of amigrating column and then organize to form new capillary tubes.

[0004] Persistent, unregulated angiogenesis occurs in a multiplicity ofpathological conditions, tumor metastasis and abnormal growth byendothelial cells and supports the pathological damage seen in theseconditions. The diverse pathological disease states in which unregulatedangiogenesis is present have been grouped together as angiogenicdependent or angiogenic associated diseases. Outgrowth of new bloodvessels under pathological conditions can lead to the development andprogression of diseases such as tumor growth, diabetic retinopathy,tissue and organ malformation, obesity, macular degeneration, rheumatoidarthritis, and cardiovascular disorders.

[0005] Several studies have produced direct and indirect evidence ofproof that tumor growth and metastasis are angiogenesis-dependent(Brooks et al., Cell, 1994, 79, 1154-1164; Kim K J et al., Nature, 1993,362, 841-844). Expansion of the tumor volume requires the induction ofnew capillary blood vessels. Tumor cells promote angiogenesis by thesecretion of angiogenic factors, in particular basic fibroblast growthfactor (bFGF) (Kandel J. et al., Cell, 1991, 66, 1095-1104) and vascularendothelial growth factor (VEGF) (Ferrara et al., Endocr. Rev., 1997,18: 4-25). Tumors may produce one or more of these angiogenic peptidesthat can synergistically stimulate tumor angiogenesis (Mustonen et al.,J Cell Biol., 1995, 129, 865-898). Therefore, expression oradministration of anti-angiogenic factors by gene therapy, for instance,should counteract the tumor-induced angiogenesis.

[0006] Various anti-angiogenic polypeptides have been discussed and usedto treat human angiogenic dependent or angiogenic associated diseases.For example, angiostatin and endostatin, which are proteolytic fragmentsof plasminogen (Pgn) and collagen XVIII, respectively (O'Reilly et al.,Cell, 1994, 79:315-328; O'Reilly et al., Cell, 1997, 88:1-20).Angiostatin contains the first four disulfide-linked structures ofplasminogen, which are known as kringle structures, and which displaydifferential effects on the suppression of the endothelial cell growth.For example, kringle 1 was shown to exhibit some inhibitory activity,while kringle 4 is an ineffective fragment. Hua L et al., (BBRC, 1999,258 :668-673) has characterized another kringle structure withinplasminogen but ouside of angiostatin, g., kringle 5. The kringle 5 wasshown to inhibit endothelial cell proliferation and migration. Also,Renhai C. et al. (PNAS, 1999, Vol. 96, No. 10, pp. 5728-5733) hasdemonstrated a synergistic effect on endothelial inhibition whenangiostatin and kringle 5 were coincubated with capillary endothelialcells. It was, however, stated that such association did not completelyarrest tumor growth or tumors at a dormant stage.

[0007] The prothrombin kringle-2 domain, which is a fragment releasedfrom prothrombin by factor Xa cleavage, was identified as havinganti-endothelial cell proliferative activity by Lee T H et al. (JBC,1998, vol 273, No. 44, pp. 25505-25512) using in vitro angiogenesisassay system with bovine capillary endothelial (BCE) cell proliferation.The prothrombin kringle-2 domain was, however, described as havingendothelial cell suppression activities comparable with those ofangiostatin.

[0008] An amino terminal portion of the urokinase plasminogen activatoruPA, termed ATF, has also been disclosed (Li et al., Hum Gen Ther 10:3045-53, 1999; Griscelli et al., HumGenTher, 1999, Vol 10, No. 18, pp.3045-53) as inhibiting angiogenesis. uPA is composed of three domains, aserine proteinase domain, a kringle domain, and a growth-factor-likedomain. The urokinase plasminogen binds to its receptor (uPAR) by itsgrowth-factor-like domain, and initiates a proteolytic cascade at thesurface of migrating cells to stimulate intracellular signalingresponsible for cell migration and proliferation. The uPA lacking thegrowth-factor-like domain was, however, unable to associate with uPARand was rapidly cleared from the cell surface (Poliakov et al., BiochemJ., 2001, 355:639-45).

[0009] Binding of uPA to its receptor greatly potentiatesplasminogen/plasmin conversion at the cell surface. Plasmin is a broadlyspecific serine protease, which can directly degrade components of theextracellular matrix. uPA and plasmin are somehow involved in cellmorphogenesis by activating or inducing the release of morphogenicfactors, such as vascular endothelial growth factor (VEGF), hepatocytegrowth factor (HGF), or fibroblast growth factor (FGF). Clinicalobservations correlate the presence of enhanced uPA activity at theinvasive edge of the tumors (Schmitt M et al., Fibrinolysis, 1992, 6,3-26). ATF is capable of mediating disruption of the uPA/uPAR complexand inhibiting tumor cell migration and invasion in vitro (H. Lu et al.,FEBS Letter, 1994, 356, 56-59). However, the ATF molecule retains theEGF growth factor binding domain, which interacts with the uPARreceptor. Such interactions may facilitate tumor growth, as suggested inthe scientific literature (Rabbani et al., J Biol. Chem 275:16450-58(1992)).

SUMMARY OF THE INVENTION

[0010] The present invention provides kringle-containing polypeptides,called abrogens, that are potent inhibitors of endothelial proliferationand angiogenesis. The abrogen polypeptides are capable of inhibiting orreducing cell proliferation induced by both bFGF and VEGF in a specificendothelial cell proliferation assay, whereas angiostatin only inhibitsbFGF induced proliferation in this assay. Furthermore, vectors thatexpress abrogen polypeptides in vivo reduce tumor metastasis in two lungcancer models. Thus, aspects of the invention include novelpolypeptides, nucleic acids that encode them, vectors containing them,and methods of using any of these aspects to express polypeptides, altergrowth or other characteristics of cells, or treat or prevent diseaseare provided by the invention.

[0011] Embodiments of the abrogen activity include a region of urokinaseplasminogen activator encompassing the kringle domain. The mammalianurokinase plasminogen activator (uPA) kringle domain (ATF-kringle) hasnot been previously identified as a separate molecule withanti-angiogenic activity. Rather, it was previously shown to be a potentsource of attraction of smooth muscle cells [2]. Surprisingly, weidentify and show that the ATF-kringle retains a very potentanti-angiogenic activity, while not containing the growth-factor-likedomain acting as binding site to the uPAR, thereby allowing uPA/uPARcomplex disruption. As demonstrated in Example 3, for example,ATF-kringle containing polypeptides can inhibit endothelial cellactivation and/or proliferation mediated by several differentproangiogenic proteins, such as bFGF and VEGF, in a species independentmanner.

[0012] The use of the kringle domain allows greater specificity in theanti-angiogenic mode of action. Our data from in vitro studies showsthat the ATF-kringle molecule possesses a new activity that inhibitsboth bFGF and VEGF induced tube formation and/or cell proliferation in aspecific endothelial cell assay. This assay also distinguishes thespecies-specific activity of other anti-angiogenic polypeptides. Theabrogen polypeptides, and in particular those of SEQ ID No.: 1, 3, 5,and 7, do not show a species-specific response and both mouse and humanderived polypeptides, for example, function in a mouse model system.This can be advantageous in developing human therapeutic compositionsbased upon a mouse model system. In another contrast over previouspolypeptides, anti-angiogenic factors such as endostatin or angiostatinonly inhibit bFGF-induced activity in this assay (Chen et al., Hum GenTher 11: 1983-96 (2000)). In general terms, the invention encompassesthe production of, identification of, and use of polypeptides, as wellas the nucleic acids that encode them, that possess this new activity,referred to as abrogens.

[0013] Thus, in one aspect, the invention comprises an isolated abrogenpolypeptide, such as one with an amino acid sequence of SEQ ID NO.: 1,3, 5, or 7 the polypeptide being in a form that does not exist in natureand has not been previously disclosed. The abrogen polypeptide can be inpurified form, so that, for example, it is no longer inside a cell thatproduces it, it is in an extract derived from a cell that produces it,it is at least partially separated from a final reaction mixture thatproduces it, or one or more components of a mixture containing it havebeen substantially or to a measurable extent removed. A purified formcan also be a form suitable for pharmaceutical research use, such as aform substantially free of antigenic or inflammatory components. Apurified form can also be the result of an affinity purificationprocess.

[0014] The invention also includes a nucleic acid comprising orconsisting of a sequence that encodes an abrogen polypeptide, such asthe sequences of SEQ ID NO.: 2, 4, 6, or 8. The nucleic acid can be DNA,RNA, or DNA or DNA comprising modified nucleotide bases. A nucleic acidencoding an abrogen polypeptide can also be operably linked to a varietyof one or more sequences used in expression vectors, and/or cloningvectors, and/or other vectors. For example, the abrogen encoding nucleicacid can be linked to a promoter, enhancer, a sequence encoding a signalsequence, and/or a sequence encoding an affinity purification sequence.One of ordinary skill in the art is familiar with selecting appropriatesequence(s) or vector(s) and using them. The invention also encompassescells that contain or comprise an abrogen polypeptide or abrogenencoding nucleic acid.

[0015] The cell can be transduced with, transfected with, or have anintroduced into it a vector that comprises the abrogen encoding nucleicacid. Progeny of the cell, for example cells that result from culturedcell splitting or maintenance procedures, are also included in theinvention. The cell can be a cultured primary cell, an established cellline cell, a transformed cell, a tumor cell, an endothelial cell, or avariety of other mammalian cells.

[0016] The invention also comprises a novel purified polypeptide thatcomprises a fragment of a mammalian or human kringle-containing protein,the fragment having a kringle domain that is capable of inhibiting tubeformation in endothelial cell cultures induced by bFGF and VEGF, and/orcapable of reducing cell proliferation induced by bFGF and VEGF, and/orcapable of inhibiting metastasis of mammalian tumors. This fragment doesnot contain an EGF-binding domain, such as the EGF-binding domain of uPAor the amino terminal fragment (ATF) of uPA. The novel purifiedpolypeptide does not contain the exact amino acid sequence of thekringle 5 domain of human plasminogen, the exact sequence of kringle 2from human prothrombin, the exact 80 amino acids beginning at residue462 of human plasminogen, or the exact sequence of any of the previouslydisclosed kringle-containing polypeptides, peptides, or proteins. Thenovel polypeptides can advantageously be used in a number of instanceswhere inhibiting or reducing cell proliferation associated with bFGF andVEGF treatment is desired, and/or where inhibiting angiogenesis or tumormetastasis is desired.

[0017] In another aspect, the invention comprises nucleic acids thatencode these novel polypeptides, vectors containing them, and cellscontaining them. Preferably, inhibiting tube formation in endothelialcell cultures induced by bFGF and VEGF, reducing cell proliferationinduced by bFGF and VEGF, and/or inhibiting metastasis of mammaliantumors is measured in culture with established endothelial cell lines ortumor cell lines. However, other types of measurements, includingmeasurements in vivo, can also be used. In this and other aspects of theinvention involving cells, a preferred embodiment employs or involveshuman umbilical vein endothelial cells or mammary or lung tumor cells.

[0018] Preferably, the kringle-containing protein is human protein, suchas a human plasminogen activator, like urokinase plasminogen activatoror tissue plasminogen activator. Other human proteins from which thenovel polypeptides and nucleic acids of the invention can be derived areApoArgC, Factor XII, hepatocyte growth factor activator, hyaluronanbinding protein, macrophage stimulating protein, thrombin, retinoic acidreceptors 1 and 2, and kringle containing domains from extended sequencetag database or other database. In preferred examples, thesepolypeptides comprise a kringle domain having a region of SEQ ID NO.: 1from Asn 53 to Asp 59 [NYCRNPD], and further comprises one or moreregions within a particular amino acid sequence identity range to aregion of SEQ ID NO.: 1, 3, 5, or 7. In particular, the regions of SEQID NO.: 1 that may be modified include from Cys 3 to Trp 27, Asn 53 toCys 84, Lys 1 to Thr 2, and Ala 85 to Asp 86. However, these derivativescontain the conserved 6 Cys residues that are thought to help properlyfold the kringle domain into a characteristic structure. Various regionsare quite amenable to modification by substitution, deletion, and/oraddition, including the region from about Asn 28 to about His 52 or Lys51, and the terminal 2 residues from each of the N terminus andC-terminus of SEQ ID NO.: 1. Particularly preferred derivatives includethose with a region of approximately 50% amino acid identity to theregion of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region ofapproximately 40% amino acid identity to the region of SEQ ID NO.: 1from Asn 53 to Cys 84; a region of approximately 55% amino acid identityto the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region ofapproximately 45% amino acid identity to the region of SEQ ID NO.: 1from Asn 53 to Cys 84; a region of approximately 35% amino acid identityto the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region ofapproximately 35% amino acid identity to the region of SEQ ID NO.: 1from Asn 53 to Cys 84. For each of the abrogen regions identified hereor elsewhere in this disclosure, one of skill in the art can clearlyselect an optimum or desirable range or specific sequence identitydifference from that listed in the previous sentence. Thus, the 50%percent amino acid identity noted here and elsewhere can also be 55%, or60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, orfrom about 50-55%, or 55-60%, or 60-65%, or 65-70%, or 70-75%, or75-80%, or 80%-85%, or 85%-90%, or 90-95%, or 95-98%, or 98-99%.Similarly, the 40% noted here or elsewhere can be 45%, 50%, and aboveand in various ranges as just listed, and the 35% noted here andelsewhere can be 40%, or 45% and above and in various ranges as justlisted. Additional examples include an abrogen polypeptide with aminoacid sequence of SEQ ID NO.: 1 modified to contain 1 to about 15 aminoacid changes of substitutions, deletions, or additions, wherein theamino acid changes occur in the amino acids from Asn 28 to His 52, Lys 1to Thr 2, Ala 85 to Asp 86. Furthermore, derivatives may merely containor may additionally contain 1 to about 5, 1 to about 10, 1 to about 15,or 1 to about 20 amino acid changes outside of the consensus region fromAsn 53 to Asp 59 of SEQ ID NO.: 1 [NYCRNPD] that are conservative aminoacid substitutions.

[0019] The polypeptides and the nucleic acids that encode them mayadditionally have or encode a selected signal sequence region and/or anaffinity purification sequence region. As used herein, the term “signalsequence or signal peptide” is understood to mean a peptide segmentwhich directs the secretion of the abrogen polypeptides or abrogenfusion polypeptides and thereafter is cleaved following translation inthe host cells. The signal sequence or signal peptide thus initiatestransport of a protein across the membrane of the endoplasmic reticulum.Signal sequences have been well characterized in the art and are knowntypically to contain 16 to 30 amino acid residues, and may containgreater or fewer amino acid residues. A typical signal peptide consistsof three regions: a basic N-terminal region, a central hydrophobicregion, and a more polar C-terminal region. The central hydrophobicregion contains 4 to 12 hydrophobic residues that anchor the signalpeptide across the membrane lipid bilayer during transport of thenascent polypeptide. Following initiation, the signal peptide is usuallycleaved within the lumen of the endoplasmic reticulum by cellularenzymes known as signal peptidases (von Heijne (1986) Nucleic AcidsRes., 14: 4683). Numerous examples exist including the well knownpoly-His tag sequence, the immunoglobulin signal sequence, and the humaninterleukin 2 (IL2) signal sequence.

[0020] The polypeptide and the sequence encoding the polypeptide used ina specific vector encoding the given kringle domain may also be linkedto stabilizing elements or polypeptides or the sequences that encodethem, such as those from human serum albumin or the immunoglobulin Fcportion of an IgG molecule.

[0021] The abrogen polypeptides according to the present invention maybe advantageously linked to a human serum albumin (HSA) or other fusionpartner. Such fusion polypeptides comprise the abrogen polypeptide fusedat its C- or N-terminal with HSA. The amino acid sequence of HSA is wellknown in the art and is inter alia disclosed by Meloun et al. (CompleteAmino Acid Sequence of HSA, FEBS Letter: 58:1. 136-137, 1975) andBehrens et al. (Structure of HSA, Fed. Proc. 34,591, 1975), and morerecently by genetic analysis (Lawn et al., Nucleic Acids Research, 1981,9, 6102-6114). Shorter forms or variants of HSA, as described in EP 322094, may also be used to produce the abrogen fusion protein of theinvention. Any abrogen polypeptide noted here can be used to prepare anabrogen fusion protein or polypeptide of the invention. Construction ofsuch fusion proteins is well known in the art and is disclosed interalia, in U.S. Pat. No. 5,876,969. Fusion proteins so obtained possess aparticularly advantageous distribution in the body, while modifying thepharmacokinetic properties of the abrogen poplypeptide and compositionscontaining them, and favors the development of their biologicalactivity.

[0022] An abrogen fusion protein or polypeptide according to the presentinvention may also comprise an N-terminal signal peptide, such as theIL2 signal peptide providing for secretion into the surrounding medium,followed or preceded by a HSA or a portion thereof, or a variant thereofand the sequence of the abrogen polypeptides. The abrogen polypeptidesmay be coupled either directly or via an artificial peptide or linker toalbumin, at the N-terminal end or the C-terminal end.

[0023] The chimeric molecule may be produced by eucaryotic, prokaryotic,or cellular hosts that contain a nucleotide sequence encoding theabrogen fusion protein, and then harvesting the polypeptide produced.Animal cells, yeast, fungi may be used as eucaryotic hosts. Inparticular, yeasts of the genus of Saccharomyces, Kluveromyces, Pichia,Schwanniomyces, or Hansenula may be cited. Animal cells, such as forexample, COS, CHO, 293 cell lines, and C127 cells, and the like may beused. Fungi such as Aspergillus sp., or Trichoderma ssp may be used.Bacteria, such as Esherichia coli, or bacteria belonging to the generaof Corynebacterium, Bacillus, or Streptomyces may be used as prokaryoticcells.

[0024] In another fusion protein or polypeptide example, the abrogenpolypeptide is fused to an immunoglobulin Fc region as described in WO00/01133. Immunoglobulin Fc region is understood to mean thecarboxylterminal portion of an immunoglobulin chain constant region,preferably an immunoglobulin heavy chain constant region, or a portionthereof. For example, an immunoglobulin Fc region may comprise: 1) animmunoglobulin constant heavy 1 (CH1) domain, an immunoglobulin constantheavy 2 (CH2) domain, and an immunoglobulin constant heavy (CH3) domain;2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) aCH2 domain and a CH3 domain; and/or 5) a combination of two or moredomains and an immunoglobulin hinge region. In a preferred embodimentthe Fe region used in the DNA construct encoding the abrogen polypeptidealso encodes an immunoglobulin hinge region, CH2 and CH3 domains, anddepending upon the type of immunoglobulin used to generate the Fcregion, optionally a CH4 domain. More preferably, the immunoglobulin Fcregion comprises a hinge region, and CH2 and CH3 domains. Immunoglobulinfrom which the heavy chain constant region is preferably derived is IgGof subclasses 1, 2, 3, or 4, and most preferably of subclass 2, mostpreferably the murin or human immunoglobulin Fc region from IgG2a. Otherclasses of immunoglobulin, IgA, IgD, IgE and IgM, may be used. Thechoice of appropriate or advantageous immunoglobulin heavy chainconstant regions is discussed in detail in U.S. Pat. Nos. 5,541,087, and5,726,044. The choice of particular immunoglobulin heavy chain constantregion sequences from certain immunoglobulin classes and subclasses toachieve a particular result is considered to be within the level ofskill in the art. The Fe region used in the fusion protein is preferablyfrom a mammalian species, for example from murine origin, and preferablyfrom human origin, or from a humanized Fe region.

[0025] The fusion proteins of the invention preferably are generated byconventional recombinant DNA methodologies. The fusion proteinspreferably are produced by expression in a host cell of a DNA moleculeencoding a signal sequence, an immunoglobulin Fe region or HSA forexample, and an abrogen polypeptide. The constructs may encode in a 5′to 3′ direction, the signal sequence, the immunoglobulin Fe region orHSA for example, and the abrogen polypeptide. Alternatively, theconstructs may encode in a 5′ to ₃′ direction, the signal sequence, theabrogen polypeptide and the immunoglobulin Fe region or HSA for example.As noted above, other fusion partners or stabilizing elements orpolypeptides can be selected for use. The abrogen polypeptide may becoupled either directly or via a linker to the immunoglobulin Fe regionor HAS, for example. The fusion of the abrogen with the immunoglobulinFe region are produced by introducing into mammalian cell suchconstructs, and culturing the mammalian cells to produce the fusionproteins. The resulting fusion protein can be harvested, refolded ifnecessary, and purified using conventional purification techniques wellknown and used in the art. The resulting abrogen polypeptides exhibitlonger serum half-lives, presumably due to their larger molecular sizes,and other advantageous properties.

[0026] The abrogen polypeptides and either the HSA or the immunoglobulinFe region, for example, may be linked by a polypeptide linker. As usedherein the term “polypeptide linker” is understood to mean a peptidesequence that can link two proteins together or a protein and an Fcregion. The polypeptide linker preferably comprises a plurality of aminoacids such as glycine and/or serine. Preferably, the polypeptide linkercomprises a series of glycine and serine peptides about 10-15 residuesin length. See, for example, U.S. Pat. No. 5,258,698, the disclosure ofwhich is incorporated herein by reference. More preferably, the linkersequence is as set forth in SEQ ID NO: 12 or 16, or comprises an Asp-Alaor an Arg-Leu sequence. It is contemplated however, that the optimallinker length and amino acid composition may be determined by routineexperimentation.

[0027] The present invention also provides methods for producing abrogenfrom non-human species as and fusion proteins, such as with HAS and Fcregions. Non-human angiogenesis inhibitor fusion proteins are useful forpreclinical studies of angiogenesis inhibitors because efficacy andtoxicity studies of a protein drug must be performed in animal modelsystems before testing in humans. A human protein may elicit an immuneresponse in mouse, and/or exhibit different pharmacokinetics, skewingthe test results. Therefore, the equivalent mouse protein is the bestsurrogate for the human protein for testing in a mouse model.

[0028] Additionally, various promoter/enhancer and RNA transcriptstabilizing elements may be included in the vector.

[0029] In another aspect, the invention comprises methods for analyzingor identifying a polypeptide that reduces or inhibits endothelial cellproliferation induced by bFGF and VEGF, and/or reduces or inhibits tubeformation induced by bFGF and VEGF, and/or reduces or inhibits tumormetastasis. In general, the method may comprise selecting a polypeptidehaving a kringle domain from a mammalian protein, the kringle domaincomprising amino acid residues Asn 53 to Asp 59 of SEQ ID NO.: 1[NYCRNPD], the kringle domain also containing 6 Cys residues and 2 Trpresidues, and introducing the polypeptide to an endothelial cell, forexample by employing an expression vector such as a recombinantadenoviral vector, a recombinant adeno-associated viral vector, or aplasmid vector. Any method for measuring the relative inhibition oftubule formation, the relative inhibition of cell proliferation, or therelative inhibition of tumor metastasis can be employed to detect apolypeptide having the appropriate characteristic or even a combinationof characteristics. The invention specifically includes polypeptides andnucleic acids encoding these polypeptides that are identified or arecapable of being identified by these methods.

[0030] Moreover, an abrogen polypeptide and compositions comprising itmay be used as a therapeutic. The polypeptide and the method forexpressing it in a cell can be, therefore, used in methods to treat orprevent a variety of angiogenesis related diseases or conditions,including, but not limited to hemangioma, solid tumors, blood bornetumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma,pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaqueneovascularization, coronary collaterals, cerebral collaterals,arteriovenous malformations, ischemic limb angiogenesis, cornealdiseases, rubeosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, arthritis, rheumatoid arthritis, diabeticneovascularization, diabetic retinopathy, macular degeneration, woundhealing, obesity, peptic ulcer, Helicobacter related diseases,fractures, keloids, vasculogenesis, hematopoiesis, ovulation-relateddisorders, menstruation-related disorders, placentation, and cat scratchfever.

[0031] In general, the use can also be for abrogating tumor vasculaturegrowth or angiogenesis associated with a tumor. One skilled in the artis familiar with polypeptide expression and purification systems as wellas methods for administering polypeptides and vectors in appropriatepharmaceutical compositions.

[0032] In another aspect, the nucleic acids encoding an abrogenpolypeptide can be used in a gene transfer method. The examples show howrecombinant plasmid and adenoviral vectors, for example, can be used toaffect metastasis in a lung tumor model. Various gene transfer and genetherapy vectors can be used in conjunction with the nucleic acids of theinvention to either analyze the activity of an abrogen polypeptide invivo or treat, prevent, or ameliorate an angiogenesis-related disease orcondition in an animal. Preferably, the animal is human or mouse. Moreparticularly, a nucleic acid encoding an abrogen of SEQ ID NO.: 1, 3, 5,or 7 can be cloned into a vector, preferably an adenoviral vector, anadeno-associated virus (AAV), a plasmid, or other suitable viral ornon-viral vector. In one embodiment, the vector is administered to atumor bearing or non-tumor bearing animal by direct intratumoralinjection, intravenous injection, intramuscular injection,electrotransfer-mediated administration, or other suitable method. Theefficacy of the abrogen expressed from the vector can be assessed in thecontext of, for example, reduction of the primary tumor and/orabrogation of metastatic dissemination.

[0033] Accordingly, the invention comprises gene transfer methods andmethods for expressing abrogen polypeptides in a cell of an animal.These methods may comprise inserting a selected abrogen encodingsequence, such as one encoding SEQ ID NO.: 1, 3, 5, or 7, into amammalian expression vector or the expression cassette of an appropriatevector. The vector is administered to a cell of the animal by any numberof methods available, including intratumoral injection, electrotransfer,infision, subcutaneous injection, intramuscular injection, orintravenous administration. The effect of the expressed abrogenpolypeptide can then be measured and compared to control. These methodscan be used to treat any one of a number of angiogenesis relateddiseases or disorders, such as those listed above.

[0034] The invention also comprises administration of the abrogenrecombinant polypeptides in a cell of an animal. These methods maycomprise administering the abrogen peptide as in SEQ ID NO: 1, 3, 5, 7by any well-known method in the art, including for example, directinjections of the peptide at a specific site, i.e., by ophthalmic(including intravitreal or intraorbital), intraperitoneal,intramuscular, or intratumoral injections.

[0035] The invention also includes compositions comprising the abrogenpolypeptides or nucleic acids, and the derivatives and nucleic acidsencoding derivatives, such as those having the sequences of SEQ ID NO.:1-8 or SEQ ID NO.: 9, 10, 13, 14, 15, 17, 18, 20, or 21, or abrogenfusion polypeptides or nucleic acids encosing them. The abrogenpolypeptides or derivatives can be recombinant polypeptides or purifiedpolypeptides. The compositions of the present invention may be providedto an animal by any suitable means, directly (ea., locally, as byinjection, implantation or topical administration to a tissue locus) orsystemically (e.g., parenterally or orally). Where the composition is tobe provided parenterally, such as by intravenous, subcutaneous,ophthalmic (including intravitreal or intracameral), intraperitoneal,intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral,intracranial, intraspinal, intraventricular, intrathecal, intracistemal,intracapsular, intranasal or by aerosol administration, the compositionpreferably comprises part of an aqueous or physiologically compatiblefluid suspension or solution. Thus, the carrier or vehicle isphysiologically acceptable so that in addition to delivery of thedesired composition to the patient, it does not otherwise adverselyaffect the patient's electrolyte and/or volume balance. The fluid mediumfor the agent thus can comprise normal physiologic saline (e. g., 9.85%aqueous NaCl, 0.15 M, pH 7-7.4). In one embodiment, the composition is apharmaceutically acceptable composition. One skilled in the art isfamiliar with selecting and testing pharmaceutically acceptablecompositions for use with recombinant polypeptides and nucleic acids.

[0036] The abrogen formulations may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s).

[0037] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0038] It is another aspect or object of the present invention toprovide a method of treating diseases and processes that are mediated byangiogenesis.

[0039] It is yet another aspect of the present invention to provide amethod and composition for treating diseases and processes that aremediated by angiogenesis including, but not limited to, hemangioma,solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia,psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis,Crohn's disease, plaque neovascularization, coronary collaterals,cerebral collaterals, arteriovenous malformations, ischemic limbangiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabeticretinopathy, retrolental fibroplasia, arthritis, rheumatoid arthritis,diabetic neovascularization, diabetic retinopathy, macular degeneration,wound healing, peptic ulcer, Helicobacter related diseases, fractures,keloids, vasculogenesis, hematopoiesis, ovulation, menstruation,placentation, obesity and cat scratch fever.

[0040] It is another aspect of the present invention to provide acomposition for treating or repressing the growth of a cancer.

[0041] It is still another aspect of the present invention to provide amethod for treating ocular angiogenesis related diseases such as maculardegeneration or diabetic retinopathy by direct ophthalmic injections ofthe recombinant abrogen peptides.

[0042] Another aspect of the present invention is to provide a methodfor targeted delivery of abrogen compositions to specific locations.

[0043] Yet another aspect of the invention is to provide compositionsand methods useful for gene therapy for the modulation of angiogenicprocesses.

[0044] Throughout this disclosure, applicants refer to journal articles,patent documents, published references, web pages, sequence informationavailable in databases, and other sources of information. One skilled inthe art can use the entire contents of any of the cited sources ofinformation to make and use aspects of this invention. Each and everycited source of information is specifically incorporated herein byreference in its entirety. Portions of these sources may be included inthis document as allowed or required. However, the meaning of any termor phrase specifically defined or explained in this disclosure shall notbe modified by the content of any of the sources. The description andexamples that follow are merely exemplary of the scope of this inventionand content of this disclosure. One skilled in the art can devise andconstruct numerous modifications to the examples listed below withoutdeparting from the scope of this invention.

BRIEF DESCRIPTION OF THE FIGURES

[0045]FIG. 1: The proliferative response of transduced HUVEC humanendothelial cells to human abrogen (hATF-K; SEQ ID NO. 1) and mouseabrogen (mATK-K; SEQ ID NO.: 3). Cultured cells were transduced withadenoviral vectors containing an expression cassette for producing theabrogen polypeptide (hATF-K and mATF-K), a control, CMV promoter onlyvector (CMV), and the full amino terminal fragment of plasminogen (hATFor mATF). In FIG. 1A, the left axis indicates the degree of cellproliferation and each of the boxes represents the level of cellproliferation under a treatment regimen as indicated by the addition ofbFGF, VEGF, or both. The reduction in cell proliferation in all sampleswhere the human abrogen polypeptide is expressed (hATF-K) is markedlyreduced compared to controls (CMV, HATF, and mATF). The proliferation inthe mouse abrogen expressing cells (mATF-K) is also markedly reduced.Figure lB shows representative cell cultures from mouse and human fullATF polypeptides and mouse and human ATF-Kringle containing abrogenpolypeptides (see Examples). The first page shows Control (full humanATF treated with FGF) compared to hATF-Kringle containing polypeptidetreated with FGF. The remaining pages list the adenoviral vector used totransduce the cells (see Examples).

[0046]FIG. 2: Various human protein sequences having a kringle domainpossessing the consensus region from Asn 53 to Asp 59 of SEQ ID NO.: 1and the with the 6 conserved Cys, 2 conserved Trp, and conserved Gly andArg residues aligned. These proteins and homologs, isoforms, andderivatives of them, can be used in methods of the invention.

[0047]FIG. 3: Effect of anti-angiogenic polypeptides on tubule growth inendothelial cells. Because culture conditions rapidly depleteanti-angiogenic factors if they are added as a recombinant or purifiedpolypeptide, HUVECs are directly transduced with adenoviral vectors toprovide consistent protein expression and secretion for the duration ofthe assay (7-10 days). HUVECs are transduced with Adenovirus expressing:human abrogen, hATF-K (as in SEQ ID NO.: 1), mouse abrogen, mATF-K (asin SEQ ID NO.: 3), and human endostatin (FIG. 3A) or human Angiostatin(FIG. 3B). Control adenovirus containing the LacZ or no gene of interest(empty control) is also included. The transduced cells are then culturedin a 3-dimensional matrix of fibrin with recombinant VEGF or bFGF added,as indicated. Tubule formation as a marker for activation andproliferation of endothelial cells is then visualized and recorded.Tubule formation in both the bFGF and VEGF treated cells is markedlyinhibited in only the abrogen expressing cultures.

[0048]FIG. 4: Prevention of tumor metastasis in mouse 4T1 lung cancermodel. Control empty plasmid and abrogen (hATF-K or mATF-K) expressioncassette containing plasmid introduced via electrotransfer 6 days priorto injection of 4T1 tumor cells. Approximately 250,000 tumor cells areinjected subcutaneously. Fifteen days after injection, primary tumorsare removed in a surgical procedure. Lungs are harvested 35 days posttumor injection and the size and number of metastatic tumor coloniesmeasured.

[0049]FIG. 5: Prevention of tumor metastasis in mouse 4T1 lung cancermodel. Control empty plasmid compared to mATF-K expression plasmid. Theassay protocol is the same as in FIG. 4.

[0050]FIG. 6: Prevention of tumor metastasis in mouse 3LL Boston lungcancer model. Control empty plasmid compared to mATF-K expressionplasmid. The assay protocol is the same as FIG. 4, with the exceptionthat 3LL Boston cells are used.

[0051]FIG. 7: Prevention of tumor metastasis in mouse 3LL Boston lungcancer model. Control empty plasmid compared to experimental controlmEndostatin expression plasmid. The assay protocol is the same as FIG.6.

[0052]FIG. 8: Measurement of size and number of metastasis in the 4T1lung tumor model described for FIG. 4. Each spot represents the weightof the lung from each animal surveyed (C57BL/6 mice), indicating therelative size of the tumor nodules present. The left axis indicates thenumber of visible tumor nodules for each of the animals. With theexception of one animal in the HATF-K sample, the abrogen expressingvector treatment animals show a reduction in both the size and number ofmetastatic tumor nodules as compared to control. The hATF-K animals withabnormally high number of nodules were not further examined forexperimental or procedural error or expression of hATF-K. Here thecontrols are empty plasmid (Control) and an alkaline phosphataseexpressing control plasmid (mSEAP).

[0053]FIG. 9: Measurement of size and number of metastasis in the 3LLBoston lung tumor model described for FIG. 4 using the graphicalrepresentation method described for FIG. 7. Controls are the same as inFIG. 7. Again, the use of both the mouse and human abrogen expressingvectors (mATF-K and HATF-K) results in significant reduction in tumormetastasis.

[0054]FIG. 10: Measurement of size and number of metastasis in the 3LLBoston lung tumor model as described for FIG. 9. These data indicatethat treatment with mouse endostatin or angiostatin, or either mouse orhuman ATF-K, reduce the number and size of the lung metastatic nodulescompared to control treatment. The fact that both mouse and humanabrogen encoding vectors are efficacious indicates that thespecies-specific characteristics that limit the use of the endostatinand angiostatin polypeptides are not present in the abrogenpolypeptides. Furthermore, the abrogen polypeptides appear at least asefficacious as the either endostatin or angiostatin and much moreefficacious than a combined endostatin/angiostatin treatment(mEndo/mAngio).

[0055]FIG. 11: Systemic expression of mouse or human derived abrogenpolypeptides (here listed as MuPAK or HuPAK) from vector introduced intomuscle significantly reduces the formation of spontaneous lungmetastases in the 3LL-B tumor model. Systemic expression of therapeutictransgenes from the muscle is established 6 days before C57BL/6 mice areinjected with a tumorigenic dose of 3LL-B tumor cells. The primary tumoris carefully excised 15 days post cell injection. The study isterminated on day 35 and lung metastases were counted. Panel A: lungsfrom mice treated with empty expression vector; Panel B: mice treatedwith human derived ATF-Kringle abrogen expressing vector (HuPAK); andPanel C: with treated with mouse derived ATF-Kringle abrogen expressingvector (MuPAK); Panel D: graphically shows the number and size ofmetastatic nodules present as the diameter of each “bubble” representsthe lung weight.

[0056]FIG. 12: Systemic expression of mouse or human abrogen (herelisted as MuPAK or HuPAK) from muscle significantly reduces theformation of spontaneous lung metastases in the MDA-MB-435 tumor model.Systemic expression of therapeutic transgenes from the muscle isestablished 10 days after SCID/bg mice are injected with a tumorigenicdose of MDA-MB-435 (human breast adenocarcinoma tumor cells). Theprimary tumor is carefully excised when a volume of 250 to 350 mm3 isreached. The study is terminated on day 89 and lung metastases measured.Panel A: lungs from mice treated with control mSEAP; Panel B: withtreated with mouse derived ATF-Kringle abrogen expressing vector (hereMUPAK); Panel C: mice treated with human derived ATF-Kringle abrogenexpressing vector (HuPAK); and Panel D: graphically shows lungmetastases counts as noted above.

[0057]FIG. 13A: is a schematic representation of the plasmid pXL2996.

[0058]FIG. 13B: is a schematic representation of the plasmid pMB063.

[0059]FIG. 13C is a schematic representation of the plasmid pBA140.

[0060]FIG. 14: is a schematic representation of the plasmid pMB060 andfusion construct.

[0061]FIG. 15: is a schematic representation of the plasmid pMB059 andfusion construct.

[0062]FIG. 16 is a schematic representation of the plasmid pMB056 andfusion construct.

[0063]FIG. 17: is a schematic representation of the plasmid pMB055 andfusion construct.

[0064]FIG. 18: is a schematic representation of the plasmid pMB060mprepro and fusion construct.

[0065]FIG. 19: is a schematic representation of the plasmid pMB053 andfusion construct.

[0066]FIG. 20: is a schematic representation of the plasmid pMB057 andfusion construct.

[0067]FIG. 21: is a schematic representation of the plasmid pXL4128.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0068] A number of Kringle domain containing proteins and polypeptideshave been described and used in a variety of methods, includingtherapeutic methods. As shown here, a Kringle-containing abrogenpolypeptide can be identified and used to inhibit or reduce tumormetastasis, inhibit or reduce endothelial cell proliferation, and/orinhibit or reduce endothelial cell tubule formation. As an abrogenpolypeptide or nucleic acid encoding an abrogen polypeptide, specificexamples include the mouse or human derived kringle domains of uPA (SEQID NO.: 1-8). Additional examples have been mentioned and/or aredescribed below in their structure and/or method of making andidentifying. Functionally, an abrogen polypeptide can be distinguishedby the ability to inhibit tumor metastasis. A more specific set ofabrogen polypeptides include those that inhibit the endothelial cellproliferation induced by both of bFGF and VEGF, either in separateassays or together in one assay. An abrogen polypeptide can be eithersecreted or expressed inside a cell.

[0069] In making and using aspects and embodiments of this invention,one skilled in the art may employ conventional molecular biology, cellbiology, virology, microbiology, and recombinant DNA techniques.Exemplary techniques are explained fully in the literature. For example,one may rely on the following general texts to make and use theinvention: Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. and Sambrook et al., Third Edition (2001); DNA Cloning: APractical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. (1985)); TranscriptionAnd Translation, Hames & Higgins, eds. (1984); Animal Cell Culture (R I.Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press,(1986)); Gennaro et al. (eds.) Remington's Pharmaceutical Sciences, 18thedition; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M.Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. (2001), Coligan et al. (eds.), Current Protocols inImmunology, John Wiley & Sons, Inc. (2001); W. Paul et al. (eds.)Fundamental Immunology, Raven Press; E. J. Murray et al. (ed.) Methodsin Molecular Biology: Gene Transfer and Expression Protocols, The HumanaPress Inc. (1991); J. E. Celis et al., Cell Biology: A LaboratoryHandbook, Academic Press (1994); J. E. Coligan et al. (Eds.) CurrentProtocols in Protein Science, John Wiley & Sons (2001); and J. S.Bonifacino et al. (Eds.) Current Protocols in Cell Biology, John Wiley &Sons, Inc. (2001). Additional information sources are listed below orare referred to by citation number corresponding to the references atthe end of the specification.

[0070] As used herein, a “vector” means any nucleic acid or nucleicacid-bearing particle, cell, or organism capable of being used totransfer a nucleic acid into a host cell and/or used to cause theexpression of a polypeptide in a host cell. The term “vector” includesboth viral and nonviral products and means for introducing the nucleicacid into a cell. A “vector” can be used in vitro, ex vivo, or in vivo.Non-viral vectors include plasmids, cosmids, and can comprise liposomes,electrically charged lipids (cytofectins), DNA protein complexes, andbiopolymers, for example. Viral vectors include retroviruses,lentiviruses, adeno-associated virus, pox viruses, baculovirus,reoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barrviruses, and adenovirus vectors, for example. Vectors can also comprisethe entire genome sequence or recombinant genome sequence of a virus. Avector can also comprise a portion of the genome that comprises thefunctional sequences for production of a virus capable of infecting,entering, or being introduced to a cell to deliver nucleic acid therein

[0071] The abrogen derivatives of this invention include those havingone or more conservative amino acid substitutions. For example, one ormore amino acid residues within a sequence can be substituted by anotheramino acid of a similar polarity, which acts as a functional equivalentwhen the substitution results in no significant change in activity in atleast one selected biological activity or function.

[0072] Substitutions for an amino acid within the sequence may beselected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. Amino acids containing aromatic ring structures arephenylalanine, tryptophan, and tyrosine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

[0073] “Isolated,” when referring to a nucleic acid or polypeptide,means that the indicated molecule is present in the substantial absenceof at least one other molecule with which it naturally occurs ornecessarily occurs because of its method of preparation. Thus, forexample, an “isolated abrogen polypeptide” refers to a moleculesubstantially free of a macromolecule existing in a cell used to producethe abrogen polypeptide. However, the preparation or sample containingthe molecule may include other components of different types. Inaddition, “isolated from” a particular molecule may also mean that aparticular molecule is substantially absent from a preparation orsample. Varying degrees of isolation can be prepared from methods knownin the art. Similarly, a “purified” form of a molecule is at leastpartially separated from a final reaction mixture that produces it, orone or more components of a mixture containing it have beensubstantially or to a measurable extent removed. A purified form canalso be a form suitable for pharmaceutical research use, such as a formsubstantially free of antigenic or inflammatory components. A purifiedform can also be the result of an affinity purification process or anyother purification step or process.

[0074] The “derivatives” noted here can be produced using homologsequences, modifications of an existing sequence, or a combination ofthe two. The term “homolog” is used herein to refer to similar orhomologous sequences, whether or not any particular position or residueis identical to or different from the molecule similarity or homology ismeasured against. A nucleic acid or amino acid sequence alignment mayinclude spaces. Preferably, alignment is made using the consensusresidues listed in FIG. 2, or the 6 Cys residues of the kringle domain.One way of defining a homolog is through “percent identity” between twonucleic acids or two polypeptide molecules. This refers to the percentdefined by a comparison using a basic blastn or blastp or blastxalgorithm at the default setting, unless otherwise indicated (see, forexample, NCBI BLAST home page: http://www.ncbi.nlm.nih.gov/BLAST/).Aligning a Cys residue in abrogen, for example, can be performed bycomparing sequences where the first amino acid residue or codon is for aparticular Cys, or where the particular Cys residue is set at the sameposition as that of the abrogen Cys residue. For example, the blastpalgorithm was used to generate homolog sequences, as in those of FIG. 2,by selecting the Blosum62 matrix, gap costs set at Existence: 11 andExtension: 1 (the default settings when performed). Typically, thedefault setting is used unless otherwise indicated. “Homology” can bedetermined by a direct comparison of the sequence information betweentwo polypeptide molecules by aligning the sequence information and usingreadily available computer programs. Alternatively, homology can bedetermined by hybridization of polynucleotides under conditions allowingfor the formation of stable duplexes between homologous regions anddetermining of identifying double-stranded nucleic acid.

[0075] A “functional homolog” or a “functional equivalent” of a givenpolypeptide or sequence includes molecules derived from the nativepolypeptide sequence, as well as recombinantly produced or chemicallysynthesized polypeptides, which function in a manner similar to thereference molecule or achieve a similar desired result. Thus, a“functional homolog” or a “functional equivalent” of a given kringlenucleotide region includes similar regions derived from a differentspecies, nucleotide regions derived from an isoform, or from a differentcellular source, or resulting from an alternative splicing event, aswell as recombinantly produced or chemically synthesized nucleic acidsthat function in a manner similar to the reference nucleic acid regionin achieving a desired result, such as a result in a particular assay orcell characteristic.

[0076] A “recombinant” molecule is one that has undergone at least onemolecular biological manipulation, as known in the art. Typically, thismanipulation occurs in vitro but it can also occur within a cell, aswith homologous recombination. A recombinant polypeptide is one that isproduced from a recombinant DNA or nucleic acid. A “coding sequence” or“sequence that encodes” is a sequence capable of being transcribed andtranslated into a polypeptide in a cell in vitro or in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus.

[0077] A “nucleic acid” is a polymeric compound comprised of covalentlylinked nucleotides, from whatever source. Nucleic acid includespolyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both ofwhich may be single-stranded or doublestranded. DNA includes cDNA,genomic DNA, synthetic DNA, and seri-synthetic DNA. The term “nucleicacid” also captures sequences that include any of the known baseanalogues of DNA and RNA.

[0078] A cell has been “transfected” by a vector or exogenous orheterologous nucleic acid when the vector or nucleic acid has beenintroduced inside the cell. A cell has been “transformed” or“transduced” by a vector or exogenous or heterologous nucleic acid whenthe vector or nucleic acid effects a phenotypic change or detectablemodification in the cell, such as expression of a polypeptide.

[0079] As noted, the kringle-containing fragments can be selected fromor derived from any available kringle-containing protein or polypeptide.Angiostatin contains kringle domains 1-4 of plasminogen and a separatekringle 5 domain exists. In studies by various investigators, individualkringle domains 1, 2 and 3 are found to have some anti-angiogenicactivity and to abrogate the growth of tumors in mice [12], and the samewas found for the individual kringle 5 domain [13]. Kringle homologydomains are currently found in 156 different proteins. Kringle domainsin plasminogen, thrombin, and hepatocyte growth factor have been shownto be anti-angiogenic [14]. The kringle-2 domain of prothrombin wasrecently shown to have growth inhibitory activity towards basicfibroblast growth factor stimulated capillary endothelial cells [15,16]. It has also been shown that a kringle domain of hepatocyte growthfactor is also anti-angiogenic and abrogated endothelial cell growth[17]. The abrogen polypeptides and derivatives of the invention do nothave the exact same amino acid sequence of any of these previouslydiscussed polypeptides. However, one of skill in the art may use thesequence information and functionally activity information availablefrom these studies to construct abrogen polypeptides and derivatives, asknown in the art.

[0080] Another family member is that of uPA, from which the ATFpolypeptide noted above is derived. The ATF molecule still contains theEGF like growth factor domain at the N-terminus of the molecule,followed by a kringle domain. ApoArgC, Factor XII, Hepatocyte growthfactor activator, hyaluronan binding protein, macrophage stimulatingprotein (kringles 1-4), thrombin (kringles 1 and 2), tissue typeplasminogen activator (tPA) (kringles 1 and 2), retinoic acid receptors1 and 2, and kringle domains from a protein defined in an expressedsequence tag database can all be selected for use. FIG. 2 lists anadditional source of human kringle domains. A consensus kringle domaincontaining fragment is listed at the bottom of FIG. 2 and this consensussequence can be used to construct derivatives of the specific abrogenpolypeptides disclosed here. One skilled in the art is familiar withmethods of identifying further homologs or isoforms to select and use.

[0081] Viral vectors commonly used for in vivo or ex vivo targeting andtherapy procedures are DNA-based vectors and retroviral vectors. Methodsfor constructing and using viral vectors are known in the art (see,e.g., Miller and Rosman, BioTechniques 7:980-990 (1992)). Preferably,the viral vectors are replication defective or conditionally replicationdefective, that is, they are unable to replicate autonomously in thetarget cell or unable to replicate autonomously under certainconditions. In general, the genome of the replication defective viralvectors which are used within the scope of the present invention lack atleast one region which is necessary for the replication of the virus inthe infected cell. These regions can either be eliminated (in whole orin part), be rendered non-functional by any technique known to a personskilled in the art. These techniques include the total removal,substitution (by other sequences, in particular by the inserted nucleicacid), partial deletion or addition of one or more bases to an essential(for replication) region. Such techniques may be performed in vitro (onthe isolated DNA) or in situ, using the techniques of geneticmanipulation or by treatment with mutagenic agents. Preferably, thereplication defective virus retains the sequences of its genomenecessary for encapsulating the viral particles.

[0082] DNA viral vectors include an attenuated or defective DNA virus,such as but not limited to herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Molec. Cell. Neurosci. 2:320-330 (1991)), defective herpes virusvector lacking a glyco-protein L gene, or other defective herpes virusvectors (PCT Publication WO 94/21807 and WO 92/05263); an attenuatedadenovirus vector, such as the vector described by Stratford-Perricaudetet al. (J. Clin. Invest. 90:626-630 (1992); see also La Salle et al.,Science 259:988-990 (1993)); a defective adeno-associated virus vector(Samulski et al., J. Virol. 61:3096-3101 (1987); Samulski et al., J.Virol. 63:3822-3828 (1989); Lebkowski et al., Mol. Cell. Biol.8:3988-3996 (1988)); and a conditional replicative recombinant vectors(see, for example, U.S. Pat. Nos. 6,111,243, 5,972,706, and publishedPCT documents WO 00136650, WO 0024408).

[0083] Recombinant adenoviruses display many advantages for use astransgene expression systems, including a tropism for both dividing andnon-dividing cells, minimal pathogenic potential, ability to replicateto high titer for preparation of vector stocks, and the potential tocarry large inserts (see e.g., Berkner, K. L., Curr. Top. Micro.Immunol., 158:39-66 (1992); Jolly D., Cancer Gene Therapy, 1:51-64(1994)).

[0084] It is also possible to introduce the vector in vivo as a nakedDNA plasmid. Naked DNA vectors for gene therapy can be introduced intothe desired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter or eletrotransfer device (see, e.g., Wu et al.,J. Biol. Chem. 267:963-967 (1992); Wu and Wu, J. Biol. Chem.263:14621-14624 (1988); Hartmut et al., Canadian Patent Application No.2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci.USA 88:2726-2730 (1991)). Receptor-mediated DNA delivery approaches canalso be used (Curiel et al., Hum. Gene Ther. 3:147-154 (1992); Wu andWu, J. Biol. Chem. 262:4429-4432 (1987)). Naked plasmids or cosmids canbe used in a number of gene transfer protocols and these plasmids andcosmids can be used in embodiments of this invention (see, in general,Miyake et al., PNAS 93:1320-1324 (1996); U.S. Pat. No. 6,143,530; U.S.Pat. No. 6,153,597; Ding et al., Cancer Res., 61:526-31 (2001); andCrouzet et al., PNAS 94:1414-1419 (1997). Among the preferred plamidvectors are those described in WO9710343 and WO9626270. Plasmids canalso be combined with lipid compositions, pharmaceutically acceptablevehicles, and used with electrotransfer technology, as known in the art(see, for example, U.S. Pat. Nos. 6,156,338 and 6,143,729, and WO9901157and the related devices in WO9901175).

EXAMPLES

[0085] Previous studies have shown that the ATF molecule can beeffective as an anti-tumoral and anti-angiogenic molecule especiallywhen delivered by gene therapy vectors [6]. However the presence of theEGF like domain may lead to the activation of intracellular pathways inboth tumor cells [3] and endothelial cells [7]. These activities arecounterproductive to an anti-angiogenic treatment. We have assessed thepotency of the kringle domain from human and mouse uPA (ATF-kringle) byin vitro and in vivo assays for its potential as an anti-angiogenictherapeutic. The kringle domain of human uPA was previously shown to bea potent source of attraction for smooth muscle cells [2]. This activityagain is counterproductive to use as an anti-angiogenic agent.Surprisingly, our data now shows that ATF-kringle containingpolypeptides can inhibit endothelial cell activation and/orproliferation mediated by several different proangiogenic proteins, suchas basic fibroblast growth factor (bFGF) and vascular endothelial growthfactor (VEGF), and in a species independent manner. We have designatedthe name Abrogen to this activity.

Example 1

[0086] Cloning and Manipulating Abrogen Nucleic Acids.

[0087] Exemplary primary nucleotide and polypeptide structures for boththe mouse and human abrogens sequences are shown below. ktc yeg ngh fyrgka std tmg rpc lpw nsa tvl qqt yha hrs nal qlg SEQ ID NO.:1 lgk hny crnpdn rrr pwc yvq vgl kpl vqe cmv hdc ad aaaacctgct atgaggggaa tggtcacttttaccgaggaa aggccagcac tgacaccatg SEQ ID NO.:2 ggccggccct gcctgccctggaactctgcc actgtccttc agcaaacgta ccatgcccac agatctaatg ctcttcagctgggcctgggg aaacataatt actgcaggaa cccagacaac cggaggcgac cctggtgctatgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg gtgcatgact gcgcagat ktc yhgngd syr gka ntd tkg rpc law nap avl qkp yna hrp dai slg SEQ ID NO.:3 lgkkny crn pdn qkr pwc yvq igl rqf vqe cmv hdc sl aaaacctgct atcatggaaatggtgactct taccgaggaa aggccaacac tgataccaaa SEQ ID NO:4 ggtcggccctgcctggcctg gaatgcgcct gctgtccttc agaaacccta caatgcccac agacctgatgctattagcct aggcctgggg aaacacaatt actgcaggaa ccctgacaac cagaagcgaccctggtgcta tgtgcagatt ggcctaaggc agtttgtcca agaatgcatg gtgcatgactgctctctt ktc yeg ngh fyr gka std tmg rpc lpw nsa tvl qqt yha hrs dal qlgSEQ ID NO.:5 lgk hny crn pdn rrr pwc yvq vgl kpl vqe cmv hdc adaaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg SEQ IDNO:6 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccacagatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaaccggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatggtgcatgact gcgcagat ktc yeg ngh fyr gka std tmg rpc lpw nsa tvl qqt yhahrs dal qlg lgk SEQ ID NO:7 hny crn pdn rrr pwc yvq vgl kll vqe cmv hdcad aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg SEQID NO:8 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgtaccatgcccac agatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaacccagacaac cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc tgcttgtccaagagtgcatg gtgcatgact gcgcagat

[0088] Exemplary polypeptide sequences of the fusion proteins comprisingthe human abrogen having sequence of SEQ ID NO: 1 fused to the IL-2signal peptide and to human serum albumin or immunoglobulin IgG2 Feregion, as well as linker peptide sequences, are listed below.AKTCYEGNGH FYRGKASTDT MCRPCLPWNS ATVLQQTYHA HRSDALQLGL SEQ ID NO:9GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCAD AKTCYEGNGH FYRGKASTDTMGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEQ ID NO:10 GKHNYCRNPD NRRRPWCYVQVGLKPLVQEC MVHDCAD DAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQCPFEDH VKLVNEVTEFSEQ ID NO:11 AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERNECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF YAPELLFFAKRYKAAFTECC QAADKAACLL PKLDELRDEC KASSAKQRLK CASLQKFGER AFKAWAVARLSQRFPKAEFA EVSKLVTDLT KVHTECCHGD LLECADDRAD LAKYICENQD SISSKLKECCEKPLLEKSHC IAEVENDEMP ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHPDYSVVLLLRL AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLGEYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA EDYLSVVLNQLCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP KEFNAETFTF HADICTLSEKERQIKKQTAL VELVKHKPKA TKEQLKAVMD DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGLDAGGGGSGGGGSGGGGS SEQ ID NO:12 ADAHKSEVAH RFKDLGEENF KALVLIAFAQYLQQCPFEDH VKLVNEVTEF SEQ ID NO:13 AKTCVADESA ENCDKSLHTL FGDKLCTVATLRETYGEMAD CCAKQEPERN ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYLYEIARRHPYF YAPELLFFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLKCASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD LLECADDRADLAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP ADLPSLAADF VESKDVCKNYAEAKDVFLGM FLYEYARRHP DYSVVLLLRL AKTYETTLEK CCAAADPHEC YAKVFDEFKPLVEEPQNLIK QNCELFEQLG EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCKHPEAKRMPCA EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVPKEFNAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD DFAAFVEKCCKADDKETCFA EEGKKLVAAS QAALGLDAGG GGSGGGGSGG GGSKTCYEGN GHFYRGKASTDTMGRPCLPW NSATVLQQTY HAHRSNALQL GLGKHNYCRN PDNRRRPWCY VQVGLKPLVQECMVHDCAD ADAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQCPFEDH VKLVNEVTEF SEQ IDNO:14 AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN ECFLQHKDDNPNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF YAPELLFFAK RYKAAFTECCQAADKAACLL PKLDELRDEG KASSAKQRLK CASLQKFGER AFKAWAVARL SQRFPKAEFAEVSKLVTDLT KVHTECCHGD LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHCIAEVENDEMP ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRLAKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG EYKFQNALLVRYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA EDYLSVVLNQ LCVLHEKTPVSDRVTKCCTE SLVNRRPCFS ALEVDETYVP KEFNAETFTF HADICTLSEK ERQIKKQTALVELVKHKPKA TKEQLKAVMD DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGLDAKTCYEGNGHFYR GKASTDTMGR PCLPWNSATV LQQTYHAHRS NALQLGLGKH NYCRNPDNRRRPWCYVQVGL KPLVQECMVH DCAD AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHAHRSNALQLGL SEQ ID NO:15 GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCADDAHKSEVAHRFKD LGEENFKALV LIAFAQYLQQ CPFEDHVKLV NEVTEFAKTC VADESAENCDKSLHTLFGDK LCTVATLRET YGEMADCCAK QEPERNECEL QHKDDNPNLP RLVRPEVDVMCTAFHDNEET FLKKYLYEIA RRHPYFYAPE LLFFAKRYKA AFTECCQAAD KAACLLPKLDELRDEGKASS AKQRLKCASL QKFGEPAFKA WAVARLSQRF PKAEFAEVSK LVTDLTKVHTECCHGDLLEC ADDRADLAKY ICENQDSISS KLKECCEKPL LEKSHCIAEV ENDEMPADLPSLAADFVESK DVCKNYAEAK DVFLGMFLYE YARRHPDYSV VLLLRLAKTY ETTLEKCCAAADPHECYAKV FDEFKPLVEE PQNLIKQNCE LFEQLGEYKF QNALLVRYTK KVPQVSTPTLVEVSRNLGKV GSKCCKHPEA KRMPCAEDYL SVVLNQLCVL HEKTPVSDRV TKCCTESLVNRRPCFSALEV DETYVPKEFN AETETEHADI CTLSEKERQI KKQTALVELV KHKPKATKEQLKAVMDDFAA FVEKCCKADD KETCFAEEGK KLVAASQAAL CL GGGGSGGGGSGGGGS SEQ IDNO:16 AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEQ IDNO:17 GKHNYCRNPD NRRRPWCYVQ VCLKPLVQEC MVHDCADGGG GSGCGGSGGG CSDAHKSEVAHRFKDLGEEN FKALVLIAFA QYLQQCPFED HVKLVNEVTE FAKTCVADES AENCDKSLHTLFGDKLCTVA TLRETYGEMA DCCAKQEPER NECFLQHKDD NPNLPRLVRP EVDVMCTAFHDNEETFLKKY LYEIARRHPY FYAPELLEFA KRYKAAFTEC CQAADKAACL LPKLDELRDEGKASSAKQRL KCASLQKFGE RAFKAWAVAR LSQRFPKAEF AEVSKLVTDL TKVHTECCHGDLLECADDRA DLAKYICENQ DSISSKLKEC CEKFLLEKSH CIARVENDEM PADLPSLAADFVESKDVCKN YAEAKDVFLG MFLYEYARRH PDYSVVLLLR LAKTYETTLE KCCAAADPHECYAKVFDEFK PLVEEPQNLI KQNCELFEQL GEYKFQNALL VRYTKKVPQV STPTLVEVSRNLGKVGSKCC KHPEAKRMPC AEDYLSVVLN QLCVLHEKTP VSDRVTKCCT ESLVNRRPCFSALEVDETYV PKEFNAETFT FHADICTLSE KERQIKKQTA LVELVKHKPK ATKEQLKAVMDDFAAFVEKC CKADDKETCF AEEGKKLVAA SQAALGL DAHKSEVAHR FKDLGEENFKALVLIAFAQY LQQCPFEDHV KLVNEVTEFA SEQ ID NO:18 KTCVADESAE NCDKSLHTLFGDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV DVMCTAFHDNEETFLKKYLY EIARRHPYPY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGKASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK VHTECCHGDLLECADDPADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA DLPSLAADFVESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC CAAADPHECYAKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST PTLVEVSRNLGKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES LVNRRPCFSALEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDDFAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLDAGGG GSCGGGEGGG GSKTCYEGNGHFYRGKASTD TMGRPCLPWN SATVLQQTYH AHRSNALQLG LGKHNYCRNP DNRRRPWCYVQVGLKPLVQE CMVHDCADEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSED SEQ ID NO:19DPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSCKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN HHTTKSFSRTPGKARLEPRGPTI KPCPPCKCPA PNLLGGPSVF IFPPKIKDVL MISLSPIVTC SEQ ID NO:20VVVDVSEDDP DVQISWFVNN VEVHTAQTQT HREDYNSTLR VVSALPIQHQ DWMSGKEFKCKVNNKDLPAP IERTISKPKG SVRAPQVYVL PPPEEEMTKK QVTLTCMVTD EMPEDIYVEWTNNGKTELNY KNTEPVLDSD GSYFMYSKLR VEKKNWVERN SYSCSVVHEG LHNHHTTKSFSRTPGKKTCY EGNGHFYRGK ASTDTMGRPC LPWNSATVLQ QTYHAHRSNA LQLGLGKHNYCRNPDNRRRP WCYVQVGLKP LVQECMVHDC AD AKTCYEGNGH FYRGKASTDT MGRPCLPWNSATVLQQTYHA HRSNALQLGL SEQ ID NO:21 GKHNYCRNPD NRRRPWCYVQ VGLKPLVQECMVHDCADRLE PRGPTIKPCP PCKCPAPNLL GGPSVFIFPP KIKDVLMISL SPIVTCVVVDVSEDDPDVQI SWFVNNVEVH TAQTQTHRED YNSTLRVVSA LPIQHQDWMS GKEFKCKVNNKDLPAPIERT ISKPKGSVRA PQVYVLPPPE EEMTKKQVTL TCMVTDFMPE DIYVEWTNNGKTELNYKNTE PVLDSDGSYF MYSKLRVEKK NWVERNSYSC SVVHEGLHNH HTTKSFSRTP GK

[0089] The cDNA sequence can be obtained from GenBank or a number ofavailable sources. PCR based methods can be used to retrieve the cDNAfrom an appropriate library. The cDNA can then be conveniently stored ina vector such as the pGEM or pGEX vectors by standard ligation orplasmid manipulation methods. The polypeptide encoding regions are thentransferred into an appropriate, selected expression cassette or vector.Specific examples of vectors for various applications exist, includinggene therapy (Chen et al., Hum Gen Ther 11: 1983-96 (2000); MacDonald etal., Biochecm Biophys Res Comm 264:469-477 (1999); Cao et al., J BiolChem 271:29461-67 (1996); Li et al., Hum Gene Ther 10:3045-53 (1999)).For the examples that follow, the method of Soubrier et al., GeneTherapy 6:1482-1488 (1999), is used to prepare recombinant adenoviruswith E1/E3 deletion, CMV expression promotor and SV40 polyA. The plasmidvector used below contains the Amp resistance gene, the CMV promotor,the SV40 poly A sequence, and the IL-2 signal sequence for efficientsecretion. The fairly robust adenoviral system can be selected for itsability to be used in a variety of cell types, whereas the plasmidsystem is selected for its relative efficiency of vector introduction.One skilled in the art is familiar with selecting or modifying vectorswith these or other elements for use.

[0090] Once cloned and inserted into an appropriate vector, any of theabrogen encoding sequences or abrogen derivatives encoding sequences canbe assayed for specific activity related to anti-angiogenesis using theExamples below or an assay mentioned here or in the references.

[0091] In a preferred embodiment for expressing a recombinant abrogenpolypeptide, a vector comprising the coding region for human serumalbumin linked to the C-terminus of the abrogen encoding region is used(see, for example, Lu et al., FEBS Lett. 356: 56-9 (1994)). Other fusionproteins or chimeric proteins can also be used. In another embodiment ofa fusion protein, the abrogen encoding region is linked to animmunogenic peptide or polypeptide encoding region. These fusions can beused in created antibodies or monoclonal antibodies against an abrogen.Methods for preparing antibodies are well known in the art and both thepurified abrogen polypeptides and fusion of them can be used to prepareantibodies. Monoclonal antibodies can be prepared using hybridomatechnology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J.Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., pp. 563-681 (1981)). In general, such procedures involveimmunizing an animal (preferably a mouse) with polypeptide or, morepreferably, with a secreted polypeptide expressing cell. The micesplenocytes are extracted and fused with a suitable myeloma cell line,such myeloma cell line SP20, available from the ATCC. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium andthen cloned by limiting dilution as described (Wands et al.,Gastroenterology 80:225-232 (1981)). The hybridoma cells obtainedthrough such a selection are then assayed to identify clones, whichsecrete antibodies capable of binding the polypeptide. Additionalfusions can be used to ease purification of abrogen polypeptides,including poly-His tracks, constant domain of immunoglobulins (IgG), thecarboxy terminus of either Myc or Flag epitope (Kodak), andglutathione-S-transferase (GST) fusions. Plasmids for this purpose arereadily available.

[0092] A relatively simple method for preparing recombinant or purifiedabrogen polypeptide involves the baculovirus expression system or thepGEX system (Nesbit et al., Oncogene 18:6469-6476 (1999), Nesbit et al.,J of Immunol 166:6483-90 (2001)). In the baculovirus system, plasmid DNAencoding the abrogen polypeptide is cotransfected with a commerciallyavailable, linearized baculovirus DNA (BaculoGold baculovirus DNA,Pharmingen, San Diego, Calif.), using the lipofection method (Felgner etal., PNAS 84:7413-7417 (1987)). BaculoGold virus DNA and the plasmid DNAare mixed in a sterile well of a microtiter plate containing 50 ul ofserum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).10 μl Lipofectin and 90 μl Grace's medium are added, mixed and incubatedfor 15 minutes at room temperature. The transfection mixture is addeddrop-wise to Sf 9 insect cells (ATCC CRL 1711), and seeded in a 35 mmtissue culture plate with 1 ml Grace's medium without serum. The plateis then incubated for 5 hours at 27° C. The transfection solution isthen removed from the plate and 1 ml of Grace's insect mediumsupplemented with 10% fetal calf serum is added. The cells are culturedat 27° C. for four days. The cells can then be selected forappropriately transduction and assayed for the expression of abrogenpolypeptide. If a fusion polypeptide was desired, the fusion polypeptidecan be purified by known techniques and used to prepare monoclonalantibodies.

Example 2

[0093] Proliferation Analysis of Transduced HUVEC Using Alamar Blue.

[0094] A number of different assays for analyzing cell proliferation,tubule formation, cell migration, endothelial cell growth, and tumormetastasis exist. Some of them are described in the references cited.

[0095] Human umbilical vein endothelial cells (HUVEC: Clonetics, SanDiego) are seeded at 5×10⁵ cells/well of 6-well-plate in EGM-2 medium.The cells are incubated overnight at 37° C., 5% CO₂. Endothelial CellBasal Medium (EBM) and Endothelial Cell Growth Medium (EGM) areavailable (Clonetics, San Diego). The medium is aspirated off and 500 ulof ECM medium containing 100 IT/cell viruses put over cells. The cellsare incubated at 37° C. for 2 hours, then aspirated and 1.5 ml EGM-2medium is added. The cells are again incubate overnight at 37° C.

[0096] The cells are trypsinized, counted, and seeded at 2000 cell/wellof 96-well-plate in EGM-2 medium. The cells are incubated at 37° C. for3 hours. The medium is changed into 200 μl of the following medium:Control=ECM+0.5% FBS; Test 1=control medium with bFGF 10 ng/ml; Test2=control medium with VEGF 10 ng/ml; Test 3=control medium with bFGF 10ng/ml+VEGF 10 ng/ml. After changing the medium, the cells are incubatedat 37° C. for 5 days. 20 μl Alamar Blue (BioSource International) foreach well is added. Plates are incubated at 37° C. for 6 hours and thenthe OD read at 570 nm and 595 nm.

[0097] Typical results are depicted in FIG. 1. From the results of thisproliferation assay, both human and mouse ATF-K polypeptides (SEQ IDNO.: 1, 3, 5, and 7) are very effective in abrogating the proliferationof endothelial cells induced by bFGF and VEGF.

Example 3

[0098] Assay of Transduced HUVEC Embedded in Fibrin Gel.

[0099] In an assay that distinguishes the abrogen activity fromangiostatin, human umbilical vein endothelial cells (HUVEC: Clonetics,San Diego) are seeded (passage 3, growing in EGM-2 medium) at 5×10⁵cells/well of 6-well-plate in EGM-2 medium. The embedded cell assay alsoor alternatively provides data concerning the invasiveness of theendothelial cells in response to certain treatments. Endothelial celltubule formation induced by pro-angiogenic factors such as FGF and VEGF,a characteristic measured by this assay, can be directly correlated toangiogenesis. The abrogen polypeptides used here can inhibit or reduceangiogenesis by inhibiting tubule formation. The use of virallytransduced HUVEC can provide very detailed information as to the effectsthat a selected abrogen polypeptide or derivative has on primary celltypes. The potential anti-angiogenic agents are introduced bytransduction of the cells (m-ATF, h-ATF, m-ATF-K and h-ATF-K, CMV emptywas included as a control) using a recombinant human adenovirus.

[0100] adenovirus VP/ml vp/IT IT/ml cell/flask ul/flask

[0101] Ad CMV 5.85E+12 100 5.85E+10 5.00E+068.55

[0102] Ad HATF 2.76E+12 100 2.76E+10 5.00E+0618.12

[0103] Ad mATF 5.00E+12 100 5.00E+10 5.00E+0610

[0104] Ad hATF-K 2.02E+12 161 1.25E+10 5.00E+065

[0105] Ad mATF-K 5.19E+12 51 1.02E+11 5.00E+0640

[0106] The fibrin gel includes PBS (control), VEGF or bFGF. HUVEC cellsare split ½ to ⅓ the day before transduction. On the day of thetransduction, the cells are washed with PBS. 10 ml of serum free mediumcontaining 100:1 (IT: cell ratio) of virus is incubated with the HUVECfor 2 hours to transduce the cells. The medium is then removed and thecells washed with PBS and 20 ml of full HUVEC medium placed in each T150flask.

[0107] 48 hours following transduction the cells are trypsinized and theconcentration of each cell solution adjusted to 5×10⁵ cell/ml. The assayis performed in a 24 well plate. Each well is coated with 200 μl offibrinogen solution (12 mg/ml) and 8 ul of thrombin (50 U/ml). Then ineach well is added (according to the conditions):

[0108] VEGF165 (2 μl ), b-FGF(2 μl) or nothing (final [growth factor]=1ug/ml)

[0109] Thrombin (20 ul) of a 1000 U/ml solution.

[0110] 250 μl cell solution for a final concentration of 5×105 cells/ml

[0111] 250 μl of fibrinogen

[0112] Gels set in about 30 seconds. Then, 1.5 ml of medium is added ontop. Each type of infected cells was assayed with VEGF165 alone, b-FGFalone or without any growth factor other than those already present inthe medium.

[0113] After 6 days medium is removed and cells subjected to stainingwith Dif-Quick for enhanced visualization under microscopy. Fibrin plugsare fixed in 10% formalin, and then subjected to the 3 Dif Quick stainsfor 15 mins each before being rinsed in PBS and then fixed with 10%formalin again.

[0114] Representative photographs of cells are depicted in FIG. 1B.Tubules can be seen in control cells, whereas no tubules are detected inthe hATF-K and mATF-K transduced cells. Tubule formation can becorrelated with endothelial cell invasiveness, a characteristic ofangiogenic activity. Thus, the lack of tubule formation in the abrogenpolypeptide samples (human ATF Kringle and mouse ATF Kringle)demonstrate an inhibtion of endothelial cell invasiveness, correlatingto an inhibition of angiogenesis and metastasis. In the FIG. 1Bpictures, transduced HUVEC are treated with control PBS, bFGF, or VEGF,which give the following results. For CMV control: limited structure isvisible when PBS is in the fibrin gel; with VEGF there is robustproliferation showing the phenotype generated; tubules are clearlyvisible and are ubiquitous throughout the gel, some extensions are quitelong; in the presence of bFGF the response is not as robust, thestructures, which form, are long and spindle like in appearance. Forfull human ATF polypeptide: in PBS there are a considerable number ofstructures formed; the response is far more than that seen with controlCMV transduced endothelial cells, also in relation to the CMV controlthere has been a robust response with the addition of bFGF, which isdefinitely synergistic with the human ATF transduced cells in comparisonto those transduced with CMV; in the presence of VEGF there has been aconsiderable drop in the number of visible structure when compared tothe CMV transduced cells. For full mouse ATF polypeptide: regardless ofcondition there are no structures forming in any of the gels. For humanATK Kringle (abrogen of SEQ ID NO.: 1): regardless of condition thereare no structures forming in any of the gels. For mouse ATF Kringle(abrogen of SEQ ID NO.: 3): regardless of condition there are no tubulestructures forming in any of the fibrin gels.

[0115] Without limiting the scope of the invention to any particularmode of action or mechanism, applicants offer the following possibleexplanation of these results. Human ATF still has the EGF like growthfactor domain and may stimulate the growth of endothelial cells, whichare human in origin. This growth is potentiated in the presence ofubiquitous bFGF in this assay, as one of the downstream effects of bFGFis the upregulation of uPAR. This synergy is observed when cells aretransduced with human ATF in the presence of bFGF. In the absence ofbFGF, human ATF can stimulate low level uPAR and presumably inhibitsgrowth through the action of the kringle. Hence the observed decrease innumber of structures when compared to CMV control. Mouse ATF does notcross react with human uPAR. Therefore, the mode of action is mediatedthrough the kringle domain. With human and mouse ATF-K, there is nogrowth factor domain so no proliferative events can be initiated. Thisis specific to both bFGF and VEGF induced proliferative responses.

Example 4

[0116] In Vivo Expression of Abrogen Polypeptides Using AdenoviralVectors.

[0117] For in vivo documentation of the activity of abrogen, a firstexperiment involves the systemic injection iv of 1×1011 VP of hATF-Kexpressing adenovirus. Circulating levels of hATF-K as shown by Westerncan be measured. Exemplary expression levels at d4 can be between500-1000 ng/ml in either SCID or SCID/Beige mice. The 4T1 spontaneouslymetastatic breast cell line in SCID mice is used in which animals areinjected with 2×105 cells sub-cutaneously in the right flank. At d7,when tumors were 20-40 mm3, adenovirus is injected at 1×1011 vp: Tris,CMV1.0 control Ad; MATF-K; and HATF-K. A second and third ivadministration of adenovirus can be performed. Lung metastasis is thenmeasured at about day 35, as described below.

Example 5

[0118] In Vivo Expression of Abrogen Polypeptides Using Plasmid Vectors.

[0119] Two tumor models are used, employing 4T1 tumor cells and 3LLBoston tumor cells. In the assay, the anti-tumor activity of abrogenpolypeptide in the prophylactic murine Lewis lung carcinoma model,3LL-B, in C57BL/6 mice is tested. The assay is designed to assesswhether circulating levels of abrogen prevent and/or reduce theformation and growth of spontaneously formed metastases fromsubcutaneously implanted primary tumors. The tumor cells are cultured inDMEM containing 10% FCS, sodium pyruvate, nonessential amino acids,Pen-Strep, and L-Glutamine until prepared for injection using a bufferedsaline solution. The tumor cells are injected into the right flank of8-10 week old C57BL/6 or BALB/c female mice via subcutaneous injectionof a suspension of 2.5×105 tumor cells. Six days prior to tumor cellinjection, the 25 ul of the plasmid solutions (25ug DNA in Tris EDTAwith 10% glycerol) are injected into the tibialis cranialus muscle. Theinjection site is then exposed to 4 pulses (1 pulse per second) at 100mV using a square wave pulse generator (the electrotransfer method, ET).Alternatively, the electrotransfer enhancement can utilize four electricpulses of 100 V (250 V/cm) at 1 Hz with a pulse length of 20 msec. Onabout day 15 post cell injection, the primary subcutaneous tumor wassurgically removed. At day 35, the lungs are collected and tumor nodulesmeasured. Expression levels are measured on day—1, 7, and 14 relative toelectrotransfer. A control alkaline phosphatase expressing plasmid(mSEAP) is used to assay expression.

[0120] The results of one set of experiments are depicted in FIGS. 4-10.The empty expression plasmid and the mSEAP control plasmid treatmentsresulted in many lung tumor nodules. In both the 4T1 and 3LL tumormodels, the mATF-K and hATF-K abrogen polypeptides reduced the size andnumber of metastasis. The reduction in size and number is at leastequivalent to those of the known anti-angiogenic polypeptides endostatinand angiostatin (FIG. 10).

[0121] Another set of assays with 3-LL Boston cells employingelectrotransfer enhancement with four electric pulses of 100 V (250V/cm) at 1 Hz with a pulse length of 20 msec are shown in FIG. 11.Metastases were counted using a dissecting microscope. The FIG. 11pictures of the lungs show that the formation of spontaneous lungmetastases from the primary subcutaneous tumor was significantly reducedin the two therapeutic groups receiving plasmid DNA encoding eithermouse of human ATF Kringle (listed as MuPAK or HuPAK here). Lungmetastases counts as well as lung weights, reflected by the diameter ofthe “bubble” in panel C, were reduced in both treatment groups. Deliveryof plasmid DNA encoding either the murine secreted alkaline phosphatase(mSEAP) or no protein as control to the T. cranialis muscle did notresult in a significant reduction of lung metastases. Similar resultscan be obtained in the prophylactic 4T1 mammary tumor model (data notshown).

[0122] To assess the anti-tumor activity of systemically expressedabrogen polypeptides in a human breast adenocarcinoma xenograft model ofSCID/bg mice, MDA-MB-435 tumor cells are used. These cells aresignificantly less aggressive as compared to the 4T1 and 3LL-B syngeneicmouse tumor models. However, spontaneous lung metastases formation isestablished in the time frame of 35 days post subcutaneous cellinjection. Subcutaneous palpable MDA-MB-435 tumors are established byinjecting SCID/bg mice with 10⁶ tumor cells. On day 10 post injection,plasmid DNA was transferred to the Tibialis cranialis muscle usingelectrotransfer as described previously. Briefly, 25 μg of plasmid DNA(a total of 50 μg) in a 25 μl volume are injected directly into each T.cranialis muscle followed by four electric pulses of 100 V (250 V/cm) at1 Hz with a pulse length of 20 msec. The primary tumor is carefullyremoved when the volume reached between 250 and 350 mm3, i.e. on day 39or 44 post cell injections depending on the growth of the primary tumor.The study is terminated on day 89 and lungs harvested carefully andfixed in Bouin's solution. Metastases are counted using a dissectingmicroscope. FIG. 12 shows pictures of the lungs.

[0123] Lungs from mice treated with either mouse or human AFT-Kringlecontaining polypeptide, FIG. 12 panels B and C, bear significantly fewermetastases compared to the control group (panel A) treated with theplasmid encoding mSEAP. Overall lung metastases counts are significantlyreduced as shown in panel D. By the time of treatment at day 10, no lungmetastases have been formed in the lung of SCID/bg mice, so it is mostlikely that the systemic expression of abrogen from the muscle preventsthe formation and/or growth of distant lung metastases from the primarysubcutaneous tumor. This demonstrates an inhibition of angiogenesis, ahallmark for the growth of metastatic tumors.

Example 6

[0124] Production of Derivative Abrogen Polypeptides by PCR BasedSite-Directed Mutagenesis.

[0125] In one method for generating an abrogen derivative, fouroligonucleotide primers are used. Two of these are primers that flankthe ends of the cDNA (SEQ ID NO.: 2, 4, 6, or 8 ) and contain convenientrestriction sites for cloning into a desired vector. The other twomutagenic primers are complementary and contain the mutation(s) ofinterest. Typically, the mutagenic primers overlap by about 24 basepairs. Two separate PCR reactions are performed, each using a differentoutside primer and a different mutagenic primer that anneal to oppositestrands of the DNA template. The amplified product from both PCRreactions are purified and added to a new primeness PCR mix.

[0126] After a few PCR cycles, the two products are annealed andextended at the region of overlap yielding the derivative product. Thetwo outside primers are then added to this mixture to amplify the cDNAproduct by PCR. This method can be used to introduce amino acidsubstitutions at any point in an abrogen sequence.

[0127] In addition to the conservative amino acid substitutions notedthroughout the disclosure, one skilled in the art is familiar withnumerous methods for analyzing and selecting homologs and derivativesequences to use as abrogen sequences. For example, the sequenceidentified as “Putative-K1 (Est)” in FIG. 2 can be identified bysearching for homologs using GenBank, an EST database, or any cDNA orgenomic DNA database available. The EST can be pulled from a library,PCR amplified using primers specific for the EST, or synthesized usingautomated methods. Once isolated, the polypeptide encoding region can becloned into an appropriate vector and tested as described above.

Example 7

[0128] Construction of IL2sp-abrogen Polypeptide

[0129] The combined techniques of site-directed mutagenesis and PCRamplification allowed to construct a chimeric gene encoding a chimericpeptide resulting from the translational coupling between the first 20amino acids of the interleukin 2 signal peptide, which represent asignal sequence or signal peptide that is cleaved to produce the maturefactor (Tadatsugu, T. et al. (1983) Nature 302:305) and the abrogensequences as set forth in SEQ ID NO: 4 (IL2sp-abrogen). These hybridgenes were preferably bordered in 5′ of the translational initiator ATGand in 3′ of the translational stop codon and encode chimeric proteinsof the IL2sp-abrogen. The hybrid gene is cloned in the pXL2996 (FIG.13A), under the control of the human CMV Enhancer/promoter (−522/+72)and upstream of a SV40 late poly A signal. The resulting plasmid pMB063as described in FIG. 13A was obtained. The abrogen peptide secreted fromthe plasmid pMB063 retained an alanine from the IL-2 signal peptide atthe N-terminus, and thus contains a 87 amino acid sequence as set forthin SEQ ID NO: 9.

[0130] The hybrid nucleotide sequence comprising the interleukine 2signal peptide sequence and the abrogen sequence as set forth in SEQ IDNO: 2 was cloned in plasmid pXL 2996 downstream of the human CMVenhancer/promoter (−522/+72) and upstream of a SV40 late poly A signal.The resulting plasmid pBA140 as described in FIG. 13B was obtained. Theabrogen peptide secreted from the plasmid pBA140 also retained analanine from the IL-2 signal peptide at the N-terminus, and thuscontains a 87 amino acid sequence as set forth in SEQ ID NO: 10.

EXAMPLE 8

[0131] Construction of Fusion Proteins of Abrogen and HSA

[0132] A nucleotide fragment containing from 5′ to 3′ the IL-2 signalpeptide, the nucleotide sequence encoding the human HSA as set forth inSEQ ID NO: 11, a linker, and the abrogen sequence as set forth in SEQ IDNO: 2 was cloned in plasmid pXL2996 downstream to the human CMV promoterand upstream of a SV40 polyA. The linker DA(G₄S)₃ was used (SEQ ID NO:12). The construct of the fusion protein IL2sp-HSA-linker-abrogen andthe resulting plasmid designated pMB060 are shown in FIG. 14. The fusionprotein HSA/abrogen secreted from the plasmid pMB060 has the sequence asset forth in SEQ ID NO: 13.

[0133] Another linker DA (Asp-Ala) was used. The chimeric construct ofthe fusion protein IL2sp-HSA-DA linker-abrogen and the resulting plasmidis designated pMB059 are displayed in FIG. 15. The fusion proteinHSA/abrogen secreted from the plasmid pMB059 has the sequence as setforth in SEQ ID NO: 14.

[0134] A nucleotide fragment containing from 5′ to 3′ the IL-2 signalpeptide, the abrogen nucleotide sequence as set forth in SEQ ID NO: 2,and the sequence of the human HSA (SEQ ID NO: 11), was cloned in pXL2996downstream to the human CMV promoter and upstream of a SV40 polyA. Theresulting plasmid is designated pMB056 and construct are displayed inFIG. 16. The fusion protein HSA/abrogen secreted from the plasmid pMB056has the sequence as set forth in SEQ ID NO: 15.

[0135] A nucleotide fragment containing from 5′ to 3′ the IL-2 signalpeptide, the abrogen nucleotide sequence having the sequence as setforth in SEQ ID NO: 2, a (G₄S)₃ linker (as set forth in SEQ ID NO: 16)and the sequence of the human HSA, was cloned downstream to the humanCMV promoter and upstream of a SV40 polyA. The chimeric construct of thefusion protein IL2sp-abrogen-linker-HSA and the resulting plasmiddesignated pMB055 are displayed in FIG. 17. The fusion proteinabrogen/HSA secreted from the plasmid pMB055 has the sequence as setforth in SEQ ID NO: 17.

[0136] Alternatively, a nucleotide sequence containing from 5′ to 3′ theprepro signal of HSA, the human HSA, a sequence encoding a DA(G₄S)₃linker and the abrogen nucleotide sequence as set forth in SEQ ID NO: 2was cloned in the plasmid pXL2996 downstream to the human CMV promoterand upstream of a SV40 polyA. The resulting plasmid is designatedpMB060m and the fusion protein prepro HSA—human HSA—DA(G₄S)₃linker-abrogen are displayed in FIG. 18. The fusion protein HSA/abrogensecreted from the plasmid pMB060m has the sequence as set forth in SEQID NO: 18.

Example 10

[0137] Construction of Fusion Proteins of Abrogen and IgG2a

[0138] A nucleotide fragment containing from 5′ to 3′ the IL-2 signalpeptide, the murin IgG2a Fc region (SEQ ID NO: 19) and the human abrogennucleotide sequence having the sequence as set forth in SEQ ID NO: 2 wascloned in pXL2996 downstream to the human CMV promoter and upstream of aSV40 polyA. The resulting plasmid is designated pMB053 and the fusionconstruct are displayed in FIG. 19. The fusion protein IgG2a/abrogensecreted from the plasmid pMB053 has the sequence as set forth in SEQ IDNO: 20.

[0139] A nucleotide fragment containing from 5′ to 3′ the IL-2 signalpeptide, the human abrogen nucleotide sequence having the sequence asset forth in SEQ ID NO: 2, the nucleotide sequence coding for a RL(Arginine-Leucine) linker, the murin (mu) IgG2a Fe region was cloned inpXL2996 downstream to the human CMV promoter and upstream of a SV40polyA. The resulting plasmid is designated pMB057 and the fusionconstruct are shown in FIG. 20. The fusion protein abrogen/IgG2asecreted from the plasmid pMB057 has the sequence as set forth in SEQ IDNO: 21.

Example 11

[0140] Construction of Plasmids Suitable for the Production ofRecombinant Abrogen or Fusion Polypeptide

[0141] The plasmid pXL4128, which is represented in FIG. 21 andcomprises the bacteriophage T7 promoter was also constructed, and issuitable for the production of the abrogen peptide in E coli. Suchplasmid for the production in E.coli are also described in U.S. Pat. No.6,143,518. The plasmid pYG404 as described in the Patent application EP361 991, which comprise the sequence encoding the prepro-HSA gene may beused. For example, the C-terminal of HSA is coupled in transitionalphase with a linker sequence and the abrogen nucleotide sequence. Theresulting plasmid is used for production of the peptide in yeasts, forexample.

[0142] References:

[0143] The references cited below may be referred to above by thereference number. Each of the references is specifically incorporateherein by reference.

[0144] 1. Andreasen, P. A., et al., The urokinase-type plasminogenactivator system in cancer metastasis: a review. Int J Cancer, 1997.72(1): p. 1-22.

[0145] 2. Mukhina, S., et al., The chemotactic action of urokinase onsmooth muscle cells is dependent on its kringle domain. Characterizationof interactions and contribution to chemotaxis. J Biol Chem, 2000.275(22): p. 16450-8.

[0146] 3. Rabbani, S. A., et al., Structural requirements for the growthfactor activity of the amino-terminal domain of urokinase. J Biol Chem,1992. 267(20): p. 14151-6.

[0147] 4. Quax, P. H., et al., Binding of human urokinase-typeplasminogen activator to its receptor: residues involved in speciesspecificity and binding. Arterioscler Thromb Vase Biol, 1998. 18(5): p.693-701.

[0148] 5. Min, H. Y., et al., Urokinase receptor antagonists inhibitangiogenesis and primary tumor growth in syngeneic mice. Cancer Res,1996. 56(10): p. 2428-33.

[0149] 6. Li, H., et al., Systemic delivery of antiangiogenic adenovirusAdmATF induces liver resistance to metastasis andprolongs survival ofmice. Hum Gene Ther, 1999. 10(18): p. 3045-53.

[0150] 7. Tang, H., et al., The urokinase-type plasminogen activatorreceptor mediates tyrosine phosphorylation of focal adhesion proteinsand activation of mitogenactivated protein kinase in culturedendothelial cells. J Biol Chem, 1998. 273(29): p. 18268-72.

[0151] 8. Soff, G. A., Angiostatin and angiostatin-related proteins.Cancer Metastasis Rev, 2000. 19(1-2): p. 97-107.

[0152] 9. Kleiner, D. E., Jr. and W. G. Stetler-Stevenson, Structuralbiochemistry and activation of matrix metalloproteases. Curr Opin CellBiol, 1993. 5(5): p. 891-7.

[0153] 10. Aguirre Ghiso, J. A., et al., Deregulation of the signalingpathways controlling urokinase production. Its relationship with theinvasive phenotype. Eur J Biochem, 1999. 263(2): p. 295-304.

[0154] 11. Dong, Z., et al., Macrophage-derived metalloelastase isresponsible for the generation of angiostatin in Lewis lung carcinoma.Cell, 1997. 88(6): p. 801-10.

[0155] 12. Cao, Y., et al., Kringle domains of human angiostatin.Characterization of theanti-proliferative activity on endothelial cells.J Biol Chem, 1996. 271(46): p. 29461-7.

[0156] 13. Cao, Y., et al., Kringle 5 of plasminogen is a novelinhibitor of endothelial cell growth. J Biol Chem, 1997. 272(36): p.22924-8.

[0157] 14. Nesbit, M., Abrogation of tumor vasculature using genetherapy. Cancer Metastasis Rev, 2000. 19(1-2): p. 45-9.

[0158] 15. Lee, T. H., T. Rhim, and S. S. Kim, Prothrombin kringle-2domain has a growth inhibitory activity against basic fibroblast growthfactor-stimulated capillary endothelial cells. J Biol Chem, 1998.273(44): p. 28805-12.

[0159] 16. Rhim, T. Y., et al., Human prothrombin fragment 1 and 2inhibit bFGF-induced BCE cell growth. Biochem Biophys Res Commun, 1998.252(2): p. 513-6.

[0160] 17. Xin, L., et al., Kringle 1 of human hepatocyte growth factorinhibits bovine aortic endothelial cell proliferation stimulated bybasic fibroblast growth factor and causes cell apoptosis. BiochemBiophys Res Commun, 2000.277(1): p. 186-90.

[0161] 18. Chen, C. T., et al., Antiangiogenic gene therapy for cancervia systemic administration of adenoviral vectors expressing secretableendostatin. Hum Gene Ther, 2000. 11(14): p. 1983-96.

[0162] The additional references below are also specificallyincorporated herein by reference.

[0163] Lee T -H, Rhim T, Kim S S. Prothrombin kringle-2 domain has agrowth inhibitory activity against basic fibroblast growthfactor-stimulated capillary endothelial cells. J Biol Chem 1998;273(44): 28805-28812.

[0164] Sukhatme V P. Kringle 5 causes cell cycle arrest and apoptosis ofendothelial cells. Biochem. Biophys. Res. Corn. 1999; 258: 668-673.

[0165] Cao Y, Chen A, Seong Soo A A, Richard-Weidong J, Davidson D, CaoY, Llinas M. Kringle 5 of plasminogen is a novel inhibitor ofendothelial cell growth. J Biol Chem 1997; 272(36): 22924-22928.

[0166] Sauter B V, Martinet O, Zhang W -J, Mandeli J, Woo SLC.Adenovirus-mediated gene transfer of endostatin in vivo results in highlevel of transgene expression and inhibition of tumor growth andmetastases. Proc. Natl. Acad. Sci. 2000; 97(9): 4802-4807.

[0167] Li H, Lu H, Griscelli F, Opolon P, Sun L -Q, Ragot T, Legrand Y,Belin D, Soria J, Soria C, Perricaudet M, Yeh P. Adenovirus-mediateddelivery of a uPA/uPAR antagonist suppresses angiogenesis-dependenttumor growth and dissemination in mice. Gene Therapy 1998; 5: 1105-1113.

[0168] Dong Z, Yoneda J, Kumar R, Fidler I J. Angiostatin-mediatedsuppression of cancer metastases by primary neoplasms engineered toproduce granulocyte/macrophage colony-stimulating factor. J. Exp. Med.1998; 188(4): 755-763.

[0169] Cao Y, Ji R W, Davidson D, Schaller J, Marti D, Sohndel S,McCance S G, O'Reilly MS, Llinas M, Folkmann J. Kringle domains of humanangiostatin. J. Biol. Chem. 1996; 271(56): 29461-29467.

[0170] Mukhina S, Stepanova V, Traktouev D, Poliakov A, BeabealashvillyR, Gursky Y, Minashkin M, Shevelev A, Tkachuk V. The chemotactic actionof urokinase on smooth muscle cells is dependent on its kringle domain.J. Biol. Chem. 2000; 275(22): 16450-16458.

[0171] Fischer K, Lutz V, Wilhelm O, Schmitt M, Graeff H, Heiss P,Nishiguchi T, Harbeck N, Luther T, Magdolen V, Reuning U. Urokinaseinduces proliferation of human ovarian cancer cells: characterization ofstructual elements required for growth factor function. FEBS Lett. 1998;438(1-2): 101-105.

[0172] Koopman J L, Slomp J, de Bart A C, Quax P H, Verheijen J H.Mitogenic effects of urokinse on melanoma cells are independent of highaffinity bindng to the urokinase receptor. J. Biol. Chem. 1998; 273(50):33267-33272.

[0173] Rabbani S A, Mazar A P, Bemier S M, Haq M, Bolivar I, Henkin J,Goltzman D. Structural requirements for the growth factor activity ofthe amino-terminal domain of urokinase. J. Biol. Chem. 1992; 267(20):14151-14156.

[0174]

1 27 1 86 PRT Artificial Sequence human derived abrogen 1 Lys Thr CysTyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser 1 5 10 15 Thr AspThr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val 20 25 30 Leu GlnGln Thr Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu Gly 35 40 45 Leu GlyLys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro 50 55 60 Trp CysTyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met 65 70 75 80 ValHis Asp Cys Ala Asp 85 2 258 DNA Artificial Sequence human derivedabrogen 2 aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcactgacaccatg 60 ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgtaccatgcccac 120 agatctaatg ctcttcagct gggcctgggg aaacataatt actgcaggaacccagacaac 180 cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtccaagagtgcatg 240 gtgcatgact gcgcagat 258 3 86 PRT Artificial Sequencemouse derived abrogen 3 Lys Thr Cys Tyr His Gly Asn Gly Asp Ser Tyr ArgGly Lys Ala Asn 1 5 10 15 Thr Asp Thr Lys Gly Arg Pro Cys Leu Ala TrpAsn Ala Pro Ala Val 20 25 30 Leu Gln Lys Pro Tyr Asn Ala His Arg Pro AspAla Ile Ser Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro AspAsn Gln Lys Arg Pro 50 55 60 Trp Cys Tyr Val Gln Ile Gly Leu Arg Gln PheVal Gln Glu Cys Met 65 70 75 80 Val His Asp Cys Ser Leu 85 4 258 DNAArtificial Sequence mouse derived abrogen 4 aaaacctgct atcatggaaatggtgactct taccgaggaa aggccaacac tgataccaaa 60 ggtcggccct gcctggcctggaatgcgcct gctgtccttc agaaacccta caatgcccac 120 agacctgatg ctattagcctaggcctgggg aaacacaatt actgcaggaa ccctgacaac 180 cagaagcgac cctggtgctatgtgcagatt ggcctaaggc agtttgtcca agaatgcatg 240 gtgcatgact gctctctt 2585 86 PRT Artificial Sequence human derived abrogen 5 Lys Thr Cys Tyr GluGly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser 1 5 10 15 Thr Asp Thr MetGly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val 20 25 30 Leu Gln Gln ThrTyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly 35 40 45 Leu Gly Lys HisAsn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro 50 55 60 Trp Cys Tyr ValGln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met 65 70 75 80 Val His AspCys Ala Asp 85 6 258 DNA Artificial Sequence human derived abrogen 6aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg 60ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac 120agatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac 180cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg 240gtgcatgact gcgcagat 258 7 86 PRT Artificial Sequence human derivedabrogen 7 Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys AlaSer 1 5 10 15 Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser AlaThr Val 20 25 30 Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu GlnLeu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg ArgArg Pro 50 55 60 Trp Cys Tyr Val Gln Val Gly Leu Lys Leu Leu Val Gln GluCys Met 65 70 75 80 Val His Asp Cys Ala Asp 85 8 258 DNA ArtificialSequence human derived abrogen 8 aaaacctgct atgaggggaa tggtcacttttaccgaggaa aggccagcac tgacaccatg 60 ggccggccct gcctgccctg gaactctgccactgtccttc agcaaacgta ccatgcccac 120 agatctgatg ctcttcagct gggcctggggaaacataatt actgcaggaa cccagacaac 180 cggaggcgac cctggtgcta tgtgcaggtgggcctaaagc tgcttgtcca agagtgcatg 240 gtgcatgact gcgcagat 258 9 87 PRTArtificial Sequence human derived fusion protein 9 Ala Lys Thr Cys TyrGlu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala 1 5 10 15 Ser Thr Asp ThrMet Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr 20 25 30 Val Leu Gln GlnThr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu 35 40 45 Gly Leu Gly LysHis Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg 50 55 60 Pro Trp Cys TyrVal Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys 65 70 75 80 Met Val HisAsp Cys Ala Asp 85 10 87 PRT Artificial Sequence human derived fusionprotein 10 Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly LysAla 1 5 10 15 Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn SerAla Thr 20 25 30 Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asn Ala LeuGln Leu 35 40 45 Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn ArgArg Arg 50 55 60 Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val GlnGlu Cys 65 70 75 80 Met Val His Asp Cys Ala Asp 85 11 585 PRT ArtificialSequence human derived fusion protein 11 Asp Ala His Lys Ser Glu Val AlaHis Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu ValLeu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp HisVal Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val AlaAsp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe GlyAsp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly GluMet Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys PheLeu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val ArgPro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu GluThr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His ProTyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 TyrLys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu ThrLys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu CysAla Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn GlnAsp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro LeuLeu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu MetPro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser LysAsp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe LeuGly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr SerVal Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr LeuGlu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala LysVal Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn LeuIle Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 TyrLys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr GluSer 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu ValAsp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe ThrPhe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln IleLys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro LysAla Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala AlaPhe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr CysPhe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala AlaLeu Gly Leu 580 585 12 17 PRT Artificial Sequence human derived linkerpeptide 12 Asp Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly GlyGly 1 5 10 15 Ser 13 689 PRT Artificial Sequence fusion protein humanabrogen 13 Ala Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp LeuGly 1 5 10 15 Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala GlnTyr Leu 20 25 30 Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn GluVal Thr 35 40 45 Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu AsnCys Asp 50 55 60 Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr ValAla Thr 65 70 75 80 Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys AlaLys Gln Glu 85 90 95 Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp AspAsn Pro Asn 100 105 110 Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val MetCys Thr Ala Phe 115 120 125 His Asp Asn Glu Glu Thr Phe Leu Lys Lys TyrLeu Tyr Glu Ile Ala 130 135 140 Arg Arg His Pro Tyr Phe Tyr Ala Pro GluLeu Leu Phe Phe Ala Lys 145 150 155 160 Arg Tyr Lys Ala Ala Phe Thr GluCys Cys Gln Ala Ala Asp Lys Ala 165 170 175 Ala Cys Leu Leu Pro Lys LeuAsp Glu Leu Arg Asp Glu Gly Lys Ala 180 185 190 Ser Ser Ala Lys Gln ArgLeu Lys Cys Ala Ser Leu Gln Lys Phe Gly 195 200 205 Glu Arg Ala Phe LysAla Trp Ala Val Ala Arg Leu Ser Gln Arg Phe 210 215 220 Pro Lys Ala GluPhe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr 225 230 235 240 Lys ValHis Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp 245 250 255 AspArg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile 260 265 270Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser 275 280285 His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro 290295 300 Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr305 310 315 320 Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr GluTyr Ala 325 330 335 Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu ArgLeu Ala Lys 340 345 350 Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala AlaAla Asp Pro His 355 360 365 Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe LysPro Leu Val Glu Glu 370 375 380 Pro Gln Asn Leu Ile Lys Gln Asn Cys GluLeu Phe Glu Gln Leu Gly 385 390 395 400 Glu Tyr Lys Phe Gln Asn Ala LeuLeu Val Arg Tyr Thr Lys Lys Val 405 410 415 Pro Gln Val Ser Thr Pro ThrLeu Val Glu Val Ser Arg Asn Leu Gly 420 425 430 Lys Val Gly Ser Lys CysCys Lys His Pro Glu Ala Lys Arg Met Pro 435 440 445 Cys Ala Glu Asp TyrLeu Ser Val Val Leu Asn Gln Leu Cys Val Leu 450 455 460 His Glu Lys ThrPro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 465 470 475 480 Ser LeuVal Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 485 490 495 ThrTyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 500 505 510Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 515 520525 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 530535 540 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys545 550 555 560 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly LysLys Leu 565 570 575 Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Asp Ala GlyGly Gly Gly 580 585 590 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser LysThr Cys Tyr Glu 595 600 605 Gly Asn Gly His Phe Tyr Arg Gly Lys Ala SerThr Asp Thr Met Gly 610 615 620 Arg Pro Cys Leu Pro Trp Asn Ser Ala ThrVal Leu Gln Gln Thr Tyr 625 630 635 640 His Ala His Arg Ser Asn Ala LeuGln Leu Gly Leu Gly Lys His Asn 645 650 655 Tyr Cys Arg Asn Pro Asp AsnArg Arg Arg Pro Trp Cys Tyr Val Gln 660 665 670 Val Gly Leu Lys Pro LeuVal Gln Glu Cys Met Val His Asp Cys Ala 675 680 685 Asp 14 674 PRTArtificial Sequence fusion protein human abrogen 14 Ala Asp Ala His LysSer Glu Val Ala His Arg Phe Lys Asp Leu Gly 1 5 10 15 Glu Glu Asn PheLys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu 20 25 30 Gln Gln Cys ProPhe Glu Asp His Val Lys Leu Val Asn Glu Val Thr 35 40 45 Glu Phe Ala LysThr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp 50 55 60 Lys Ser Leu HisThr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr 65 70 75 80 Leu Arg GluThr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu 85 90 95 Pro Glu ArgAsn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn 100 105 110 Leu ProArg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe 115 120 125 HisAsp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala 130 135 140Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys 145 150155 160 Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala165 170 175 Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly LysAla 180 185 190 Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln LysPhe Gly 195 200 205 Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu SerGln Arg Phe 210 215 220 Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu ValThr Asp Leu Thr 225 230 235 240 Lys Val His Thr Glu Cys Cys His Gly AspLeu Leu Glu Cys Ala Asp 245 250 255 Asp Arg Ala Asp Leu Ala Lys Tyr IleCys Glu Asn Gln Asp Ser Ile 260 265 270 Ser Ser Lys Leu Lys Glu Cys CysGlu Lys Pro Leu Leu Glu Lys Ser 275 280 285 His Cys Ile Ala Glu Val GluAsn Asp Glu Met Pro Ala Asp Leu Pro 290 295 300 Ser Leu Ala Ala Asp PheVal Glu Ser Lys Asp Val Cys Lys Asn Tyr 305 310 315 320 Ala Glu Ala LysAsp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala 325 330 335 Arg Arg HisPro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys 340 345 350 Thr TyrGlu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His 355 360 365 GluCys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu 370 375 380Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly 385 390395 400 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val405 410 415 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn LeuGly 420 425 430 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys ArgMet Pro 435 440 445 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln LeuCys Val Leu 450 455 460 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr LysCys Cys Thr Glu 465 470 475 480 Ser Leu Val Asn Arg Arg Pro Cys Phe SerAla Leu Glu Val Asp Glu 485 490 495 Thr Tyr Val Pro Lys Glu Phe Asn AlaGlu Thr Phe Thr Phe His Ala 500 505 510 Asp Ile Cys Thr Leu Ser Glu LysGlu Arg Gln Ile Lys Lys Gln Thr 515 520 525 Ala Leu Val Glu Leu Val LysHis Lys Pro Lys Ala Thr Lys Glu Gln 530 535 540 Leu Lys Ala Val Met AspAsp Phe Ala Ala Phe Val Glu Lys Cys Cys 545 550 555 560 Lys Ala Asp AspLys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 565 570 575 Val Ala AlaSer Gln Ala Ala Leu Gly Leu Asp Ala Lys Thr Cys Tyr 580 585 590 Glu GlyAsn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met 595 600 605 GlyArg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr 610 615 620Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu Gly Leu Gly Lys His 625 630635 640 Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val645 650 655 Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His AspCys 660 665 670 Ala Asp 15 672 PRT Artificial Sequence fusion proteinhuman abrogen 15 Ala Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg GlyLys Ala 1 5 10 15 Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp AsnSer Ala Thr 20 25 30 Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asn AlaLeu Gln Leu 35 40 45 Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp AsnArg Arg Arg 50 55 60 Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu ValGln Glu Cys 65 70 75 80 Met Val His Asp Cys Ala Asp Asp Ala His Lys SerGlu Val Ala His 85 90 95 Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys AlaLeu Val Leu Ile 100 105 110 Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro PheGlu Asp His Val Lys 115 120 125 Leu Val Asn Glu Val Thr Glu Phe Ala LysThr Cys Val Ala Asp Glu 130 135 140 Ser Ala Glu Asn Cys Asp Lys Ser LeuHis Thr Leu Phe Gly Asp Lys 145 150 155 160 Leu Cys Thr Val Ala Thr LeuArg Glu Thr Tyr Gly Glu Met Ala Asp 165 170 175 Cys Cys Ala Lys Gln GluPro Glu Arg Asn Glu Cys Phe Leu Gln His 180 185 190 Lys Asp Asp Asn ProAsn Leu Pro Arg Leu Val Arg Pro Glu Val Asp 195 200 205 Val Met Cys ThrAla Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys 210 215 220 Tyr Leu TyrGlu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu 225 230 235 240 LeuLeu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys 245 250 255Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu 260 265270 Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala 275280 285 Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala290 295 300 Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val SerLys 305 310 315 320 Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys CysHis Gly Asp 325 330 335 Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu AlaLys Tyr Ile Cys 340 345 350 Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu LysGlu Cys Cys Glu Lys 355 360 365 Pro Leu Leu Glu Lys Ser His Cys Ile AlaGlu Val Glu Asn Asp Glu 370 375 380 Met Pro Ala Asp Leu Pro Ser Leu AlaAla Asp Phe Val Glu Ser Lys 385 390 395 400 Asp Val Cys Lys Asn Tyr AlaGlu Ala Lys Asp Val Phe Leu Gly Met 405 410 415 Phe Leu Tyr Glu Tyr AlaArg Arg His Pro Asp Tyr Ser Val Val Leu 420 425 430 Leu Leu Arg Leu AlaLys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys 435 440 445 Ala Ala Ala AspPro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe 450 455 460 Lys Pro LeuVal Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu 465 470 475 480 LeuPhe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val 485 490 495Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 500 505510 Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro 515520 525 Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu530 535 540 Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp ArgVal 545 550 555 560 Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg ProCys Phe Ser 565 570 575 Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys GluPhe Asn Ala Glu 580 585 590 Thr Phe Thr Phe His Ala Asp Ile Cys Thr LeuSer Glu Lys Glu Arg 595 600 605 Gln Ile Lys Lys Gln Thr Ala Leu Val GluLeu Val Lys His Lys Pro 610 615 620 Lys Ala Thr Lys Glu Gln Leu Lys AlaVal Met Asp Asp Phe Ala Ala 625 630 635 640 Phe Val Glu Lys Cys Cys LysAla Asp Asp Lys Glu Thr Cys Phe Ala 645 650 655 Glu Glu Gly Lys Lys LeuVal Ala Ala Ser Gln Ala Ala Leu Gly Leu 660 665 670 16 15 PRT ArtificialSequence human derived linker peptide 16 Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Gly Gly Gly Gly Ser 1 5 10 15 17 687 PRT Artificial Sequencefusion protein human abrogen 17 Ala Lys Thr Cys Tyr Glu Gly Asn Gly HisPhe Tyr Arg Gly Lys Ala 1 5 10 15 Ser Thr Asp Thr Met Gly Arg Pro CysLeu Pro Trp Asn Ser Ala Thr 20 25 30 Val Leu Gln Gln Thr Tyr His Ala HisArg Ser Asn Ala Leu Gln Leu 35 40 45 Gly Leu Gly Lys His Asn Tyr Cys ArgAsn Pro Asp Asn Arg Arg Arg 50 55 60 Pro Trp Cys Tyr Val Gln Val Gly LeuLys Pro Leu Val Gln Glu Cys 65 70 75 80 Met Val His Asp Cys Ala Asp GlyGly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Gly Gly Gly Gly Ser Asp AlaHis Lys Ser Glu Val Ala His Arg 100 105 110 Phe Lys Asp Leu Gly Glu GluAsn Phe Lys Ala Leu Val Leu Ile Ala 115 120 125 Phe Ala Gln Tyr Leu GlnGln Cys Pro Phe Glu Asp His Val Lys Leu 130 135 140 Val Asn Glu Val ThrGlu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser 145 150 155 160 Ala Glu AsnCys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu 165 170 175 Cys ThrVal Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys 180 185 190 CysAla Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 195 200 205Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 210 215220 Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr 225230 235 240 Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro GluLeu 245 250 255 Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu CysCys Gln 260 265 270 Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu AspGlu Leu Arg 275 280 285 Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg LeuLys Cys Ala Ser 290 295 300 Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys AlaTrp Ala Val Ala Arg 305 310 315 320 Leu Ser Gln Arg Phe Pro Lys Ala GluPhe Ala Glu Val Ser Lys Leu 325 330 335 Val Thr Asp Leu Thr Lys Val HisThr Glu Cys Cys His Gly Asp Leu 340 345 350 Leu Glu Cys Ala Asp Asp ArgAla Asp Leu Ala Lys Tyr Ile Cys Glu 355 360 365 Asn Gln Asp Ser Ile SerSer Lys Leu Lys Glu Cys Cys Glu Lys Pro 370 375 380 Leu Leu Glu Lys SerHis Cys Ile Ala Glu Val Glu Asn Asp Glu Met 385 390 395 400 Pro Ala AspLeu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp 405 410 415 Val CysLys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 420 425 430 LeuTyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 435 440 445Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 450 455460 Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys 465470 475 480 Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys GluLeu 485 490 495 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu LeuVal Arg 500 505 510 Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr LeuVal Glu Val 515 520 525 Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys CysLys His Pro Glu 530 535 540 Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr LeuSer Val Val Leu Asn 545 550 555 560 Gln Leu Cys Val Leu His Glu Lys ThrPro Val Ser Asp Arg Val Thr 565 570 575 Lys Cys Cys Thr Glu Ser Leu ValAsn Arg Arg Pro Cys Phe Ser Ala 580 585 590 Leu Glu Val Asp Glu Thr TyrVal Pro Lys Glu Phe Asn Ala Glu Thr 595 600 605 Phe Thr Phe His Ala AspIle Cys Thr Leu Ser Glu Lys Glu Arg Gln 610 615 620 Ile Lys Lys Gln ThrAla Leu Val Glu Leu Val Lys His Lys Pro Lys 625 630 635 640 Ala Thr LysGlu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe 645 650 655 Val GluLys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 660 665 670 GluGly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 675 680 685 18688 PRT Artificial Sequence fusion protein human abrogen 18 Asp Ala HisLys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu AsnPhe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln CysPro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe AlaLys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser LeuHis Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 ArgGlu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 GluArg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp LysAla Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu GlyLys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu GlnLys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg LeuSer Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys LeuVal Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His GlyAsp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys TyrIle Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu CysCys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu ValGlu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala AspPhe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu AlaLys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 ArgHis Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys LysVal Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg AsnLeu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala LysArg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn GlnLeu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val ThrLys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys PheSer Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe AsnAla Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser GluLys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu ValLys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val MetAsp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala AspAsp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 AlaAla Ser Gln Ala Ala Leu Gly Leu Asp Ala Gly Gly Gly Gly Ser 580 585 590Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Thr Cys Tyr Glu Gly 595 600605 Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg 610615 620 Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr His625 630 635 640 Ala His Arg Ser Asn Ala Leu Gln Leu Gly Leu Gly Lys HisAsn Tyr 645 650 655 Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys TyrVal Gln Val 660 665 670 Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val HisAsp Cys Ala Asp 675 680 685 19 233 PRT Artificial Sequence human abrogenfusion protein IgG region 19 Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys ProPro Cys Lys Cys Pro 1 5 10 15 Ala Pro Asn Leu Leu Gly Gly Pro Ser ValPhe Ile Phe Pro Pro Lys 20 25 30 Ile Lys Asp Val Leu Met Ile Ser Leu SerPro Ile Val Thr Cys Val 35 40 45 Val Val Asp Val Ser Glu Asp Asp Pro AspVal Gln Ile Ser Trp Phe 50 55 60 Val Asn Asn Val Glu Val His Thr Ala GlnThr Gln Thr His Arg Glu 65 70 75 80 Asp Tyr Asn Ser Thr Leu Arg Val ValSer Ala Leu Pro Ile Gln His 85 90 95 Gln Asp Trp Met Ser Gly Lys Glu PheLys Cys Lys Val Asn Asn Lys 100 105 110 Asp Leu Pro Ala Pro Ile Glu ArgThr Ile Ser Lys Pro Lys Gly Ser 115 120 125 Val Arg Ala Pro Gln Val TyrVal Leu Pro Pro Pro Glu Glu Glu Met 130 135 140 Thr Lys Lys Gln Val ThrLeu Thr Cys Met Val Thr Asp Phe Met Pro 145 150 155 160 Glu Asp Ile TyrVal Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn 165 170 175 Tyr Lys AsnThr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met 180 185 190 Tyr SerLys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser 195 200 205 TyrSer Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr 210 215 220Lys Ser Phe Ser Arg Thr Pro Gly Lys 225 230 20 322 PRT ArtificialSequence fusion protein human abrogen 20 Ala Arg Leu Glu Pro Arg Gly ProThr Ile Lys Pro Cys Pro Pro Cys 1 5 10 15 Lys Cys Pro Ala Pro Asn LeuLeu Gly Gly Pro Ser Val Phe Ile Phe 20 25 30 Pro Pro Lys Ile Lys Asp ValLeu Met Ile Ser Leu Ser Pro Ile Val 35 40 45 Thr Cys Val Val Val Asp ValSer Glu Asp Asp Pro Asp Val Gln Ile 50 55 60 Ser Trp Phe Val Asn Asn ValGlu Val His Thr Ala Gln Thr Gln Thr 65 70 75 80 His Arg Glu Asp Tyr AsnSer Thr Leu Arg Val Val Ser Ala Leu Pro 85 90 95 Ile Gln His Gln Asp TrpMet Ser Gly Lys Glu Phe Lys Cys Lys Val 100 105 110 Asn Asn Lys Asp LeuPro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro 115 120 125 Lys Gly Ser ValArg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu 130 135 140 Glu Glu MetThr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp 145 150 155 160 PheMet Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr 165 170 175Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser 180 185190 Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu 195200 205 Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His210 215 220 His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys Lys Thr CysTyr 225 230 235 240 Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser ThrAsp Thr Met 245 250 255 Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr ValLeu Gln Gln Thr 260 265 270 Tyr His Ala His Arg Ser Asn Ala Leu Gln LeuGly Leu Gly Lys His 275 280 285 Asn Tyr Cys Arg Asn Pro Asp Asn Arg ArgArg Pro Trp Cys Tyr Val 290 295 300 Gln Val Gly Leu Lys Pro Leu Val GlnGlu Cys Met Val His Asp Cys 305 310 315 320 Ala Asp 21 322 PRTArtificial Sequence fusion protein human abrogen 21 Ala Lys Thr Cys TyrGlu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala 1 5 10 15 Ser Thr Asp ThrMet Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr 20 25 30 Val Leu Gln GlnThr Tyr His Ala His Arg Ser Asn Ala Leu Gln Leu 35 40 45 Gly Leu Gly LysHis Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg 50 55 60 Pro Trp Cys TyrVal Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys 65 70 75 80 Met Val HisAsp Cys Ala Asp Arg Leu Glu Pro Arg Gly Pro Thr Ile 85 90 95 Lys Pro CysPro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly 100 105 110 Pro SerVal Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile 115 120 125 SerLeu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp 130 135 140Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His 145 150155 160 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg165 170 175 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser GlyLys 180 185 190 Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala ProIle Glu 195 200 205 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala ProGln Val Tyr 210 215 220 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys LysGln Val Thr Leu 225 230 235 240 Thr Cys Met Val Thr Asp Phe Met Pro GluAsp Ile Tyr Val Glu Trp 245 250 255 Thr Asn Asn Gly Lys Thr Glu Leu AsnTyr Lys Asn Thr Glu Pro Val 260 265 270 Leu Asp Ser Asp Gly Ser Tyr PheMet Tyr Ser Lys Leu Arg Val Glu 275 280 285 Lys Lys Asn Trp Val Glu ArgAsn Ser Tyr Ser Cys Ser Val Val His 290 295 300 Glu Gly Leu His Asn HisHis Thr Thr Lys Ser Phe Ser Arg Thr Pro 305 310 315 320 Gly Lys 22 86PRT Artificial Sequence fragment of human urokinase plasminogenactivator 22 Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys AlaSer 1 5 10 15 Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser AlaThr Val 20 25 30 Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu GlnLeu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg ArgArg Pro 50 55 60 Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln GluCys Met 65 70 75 80 Val His Asp Cys Ala Asp 85 23 258 DNA ArtificialSequence fragment of human urokinase plasminogen activator 23 aaaacctgctatgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg 60 ggccggccctgcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac 120 agatctgatgctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac 180 cggaggcgaccctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg 240 gtgcatgactgcgcagat 258 24 86 PRT Artificial Sequence fragment of mouse urokinaseplasminogen activator 24 Lys Thr Cys Tyr His Gly Asn Gly Asp Ser Tyr ArgGly Lys Ala Asn 1 5 10 15 Thr Asp Thr Lys Gly Arg Pro Cys Leu Ala TrpAsn Ala Pro Ala Val 20 25 30 Leu Gln Lys Pro Tyr Asn Ala His Arg Pro AspAla Ile Ser Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro AspAsn Gln Lys Arg Pro 50 55 60 Trp Cys Tyr Val Gln Ile Gly Leu Arg Gln PheVal Gln Glu Cys Met 65 70 75 80 Val His Asp Cys Ser Leu 85 25 258 DNAArtificial Sequence fragment of mouse urokinase plasminogen activatorcDNA 25 aaaacctgct atcatggaaa tggtgactct taccgaggaa aggccaacactgataccaaa 60 ggtcggccct gcctggcctg gaatgcgcct gctgtccttc agaaaccctacaatgcccac 120 agacctgatg ctattagcct aggcctgggg aaacacaatt actgcaggaaccctgacaac 180 cagaagcgac cctggtgcta tgtgcagatt ggcctaaggc agtttgtccaagaatgcatg 240 gtgcatgact gctctctt 258 26 258 DNA Artificial Sequencefragment of human urokinase plasminogen activator 26 aaaacctgctatgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg 60 ggccggccctgcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac 120 agatctgatgctcttcngct gggcctgggg aaacataatt actgcaggaa cccagacaac 180 cggaggcgaccctggtgcta tgtgcaggtg ggcctaaagc ngcttgtcca agagtgcatg 240 gtgcatgactgcgcagat 258 27 86 PRT Artificial Sequence fragment of human urokinaseplasminogen activator 27 Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr ArgGly Lys Ala Ser 1 5 10 15 Thr Asp Thr Met Gly Arg Pro Cys Leu Pro TrpAsn Ser Ala Thr Val 20 25 30 Leu Gln Gln Thr Tyr His Ala His Arg Ser XaaAla Leu Gln Leu Gly 35 40 45 Leu Gly Lys His Asn Tyr Cys Arg Asn Pro AspAsn Arg Arg Arg Pro 50 55 60 Trp Cys Tyr Val Gln Val Gly Leu Lys Xaa LeuVal Gln Glu Cys Met 65 70 75 80 Val His Asp Cys Ala Asp 85

1. An abrogen polypeptide with an amino acid sequence consisting of SEQID NO.: 1, 3, 5, or
 7. 2. The polypeptide of claim 1 in purified form.3. A nucleic acid consisting of a sequence that encodes the polypeptideof claim 1, optionally containing a sequence encoding a signal sequenceor an affinity purification sequence.
 4. An expression vector comprisingthe nucleic acid of claim
 3. 5. A cell containing the polypeptide ofclaim 1 or progeny thereof.
 6. A cell containing the nucleic acid ofclaim 3 or progeny thereof.
 7. A purified polypeptide comprising afragment of a human protein, the fragment consisting essentially of akringle domain, wherein the polypeptide reduces cell growth induced bybFGF and VEGF.
 8. The polypeptide of claim 7, wherein the reduction incell growth is in endothelial cells.
 9. The polypeptide of claim 7,wherein the kringle domain has the amino acid sequence consisting of SEQID NO.: 1, 3, 5, or
 7. 10. The polypeptide of claim 7, wherein theplasminogen activator is urokinase plasminogen activator.
 11. A purifiedpolypeptide of claim 7, consisting of a kringle domain from a humanprotein, the kringle domain having a region of SEQ ID NO.: 1 from Asn 53to Asp 59 [NYCRNPD], the polypeptide further having a combination ofregion selected from the following group: a region of approximately 50%amino acid identity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27and a region of approximately 40% amino acid identity to the region ofSEQ ID NO.: 1 from Asn 53 to Cys 84; a region of approximately 55% aminoacid identity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and aregion of approximately 45% amino acid identity to the region of SEQ IDNO.: 1 from Asn 53 to Cys 84; a region of approximately 35% amino acididentity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and aregion of approximately 35% amino acid identity to the region of SEQ IDNO.: 1 from Asn 53 to Cys 84; wherein the polypeptide reducesendothelial cell growth induced by bFGF and VEGF.
 12. The polypeptide ofclaim 11, the polypeptide additionally having a signal sequence region.13. The polypeptide of claim 11, the polypeptide additionally having anaffinity purification sequence region.
 14. The polypeptide of claim 11,wherein the polypeptide reduces tubule formation in cultured endothelialcells.
 15. A purified nucleic acid having a sequence that encodes thepolypeptide of claim
 11. 16. An expression vector comprising the nucleicacid of claim
 15. 17. A cell comprising the vector of claim 16 orprogeny thereof.
 18. A method for identifying a polypeptide thatinhibits endothelial cell proliferation induced by bFGF and VEGF, themethod comprising selecting a polypeptide having a single kringle domainfrom a mammalian protein, the kringle domain comprising amino acidresidues Asn 53 to Asp 59 of SEQ ID NO.: 1 [NYCRNPD], the kringle domainalso containing 6 Cys residues and 2 Trp residues, introducing thepolypeptide to an endothelial cell, and measuring the inhibition oftubule formation induced by bFGF and induced by VEGF as compared to acontrol.
 19. A polypeptide identified by the method of claim
 18. 20. Anabrogen polypeptide with amino acid sequence of SEQ ID NO.: 1, 3, 5, or7, wherein 1 to about 5 amino acids outside of the consensus region fromAsn 53 to Asp 59 of SEQ ID NO.: 1 [NYCRNPD] are conservativelysubstituted for.
 21. An abrogen polypeptide with amino acid sequence ofSEQ ID NO.: 1, 3, 5, or 7 modified to contain 1 to about 15 amino acidchanges of substitutions, deletions, or additions, wherein the aminoacid changes occur in the amino acids from Asn 28 to His 52, Lys 1 toThr 2, Ala 85 to Asp 86, wherein the polypeptide inhibits endothelialcell tube formation induced by bFGF and VEGF, and wherein thepolypeptide has substantially no smooth muscle cell proliferation ormigration inducing activity.
 22. The polypeptide of claim 21, whereinthe polypeptide contains 1 to about 10 amino acid changes from SEQ IDNO.: 1, 3, 5, or
 7. 23. The polypeptide of claim 21, wherein thepolypeptide contains 1 to about 5 amino acid changes from SEQ ID NO.: 1,3, 5, or
 7. 24. The polypeptide of claim 21, further comprising 1 toabout 5 conservative amino acid substitutions outside of the consensusregion from Asn 53 to Asp 59 of SEQ ID NO.: 1 [NYCRNPD].
 25. A nucleicacid encoding the polypeptide of one of claim 18 to claim
 24. 26. Anexpression vector comprising the nucleic acid of claim
 25. 27. A cellcomprising the vector of claim 28 or progeny thereof.
 28. A method fortreating an angiogenesis related disease or disorder comprisingselecting an expression vector for expressing an abrogen polypeptide,inserting an abrogen encoding nucleic acid into the vector, andintroducing the vector.
 29. A method for treating an angiogenesisrelated disease or disorder comprising administering the abrogenpolypeptide of claim
 1. 30. The method of claim 30, wherein the disorderis tumor metastasis.
 31. The method of claim 30, wherein the vector isan adenoviral vector, an adeno associated viral vector, or a plasmidvector.
 32. The method of claim 30, wherein the abrogen encoding nucleicacid has the sequence of SEQ ID NO.: 2, 4, 6, or
 8. 33. The abrogenpolypeptide of claim 1, wherein the N-terminus of the abrogenpolypeptide is coupled to the signal peptide of interleukin
 2. 34. Theabrogen polypeptide of claim 33, wherein the abrogen polypeptide isfurther coupled to a stabilizing molecule at its C-terminus orN-terminus.
 35. The abrogen polypeptide of claim 34, wherein thestabilizing molecule is a HSA protein or a IgG2a Fe region.
 36. Theabrogen polypeptide of claim 34, wherein the C-terminus of the abrogenpolypeptide is coupled to the stabilizing molecule via a linkerpolypeptide.
 37. The abrogen polypeptide of claim 36, wherein the linkerpolypeptide has the sequence as set forth in SEQ ID NO: 9 or 10, orcomprises the amino acid sequence ARG-LEU, or ASP-ALA.